The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that α-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of α-synuclein aggregation and aggregate spreading in the context of Parkinson disease.seeding | prion-like behavior | neurodegenerative disease | kinetic analysis | electrostatic interactions T he conversion of soluble peptide and protein molecules into insoluble amyloid fibrils is of great interest in fields of science ranging from molecular medicine to nanotechnology (1). The formation of amyloid fibrils is a characteristic feature of a substantial number of increasingly common medical disorders, including neurodegenerative conditions such as Alzheimer's and Parkinson diseases (2). Elucidating the fundamental mechanistic steps involved in the conversion from the soluble to the fibrillar forms of the peptides and proteins involved in such disorders is crucial for understanding their origin and proliferation, and hence for exploring in a rational manner new and effective therapeutic strategies through which to combat their onset or progression (3).One general aspect of amyloid diseases is that once the first aggregates are formed it is very difficult to stop or reverse the aggregation process. This implies that aggregation needs to be studied in both the absence and presence of preformed aggregates, commonly known as seeds, to deepen our understanding of the mechanism of the self-assembly process in vivo. It is possible to define from in vitro studies the rate constants for the multiplicity of microscopic steps that increase the number and total mass of the different types of aggregates that are populated during this process. Such an analysis has recently been carried out for the Aβ42 peptide (4) associated with Alzheimer's disease by combining experimental and theoretical methodolo...

α-Synuclein (α-syn) is a 140-residue intrinsically disordered protein that is involved in neuronal and synaptic vesicle plasticity, but its aggregation to form amyloid fibrils is the hallmark of Parkinson's disease (PD). The interaction between α-syn and lipid surfaces is believed to be a key feature for mediation of its normal function, but under other circumstances it is able to modulate amyloid fibril formation. Using a combination of experimental and theoretical approaches, we identify the mechanism through which facile aggregation of α-syn is induced under conditions where it binds a lipid bilayer, and we show that the rate of primary nucleation can be enhanced by three orders of magnitude or more under such conditions. These results reveal the key role that membrane interactions can have in triggering conversion of α-syn from its soluble state to the aggregated state that is associated with neurodegeneration and to its associated disease states.

Intracellular α-synuclein deposits, known as Lewy bodies, have been linked to a range of neurodegenerative disorders, including Parkinson's disease. α-Synuclein binds to synthetic and biological lipids, and this interaction has been shown to play a crucial role for both α-synuclein's native function, including synaptic plasticity, and the initiation of its aggregation. Here, we describe the interplay between the lipid properties and the lipid binding and aggregation propensity of α-synuclein. In particular, we have observed that the binding of α-synuclein to model membranes is much stronger when the latter is in the fluid rather than the gel phase, and that this binding induces a segregation of the lipids into protein-poor and protein-rich populations. In addition, α-synuclein was found to aggregate at detectable rates only when interacting with membranes composed of the most soluble lipids investigated here. Overall, our results show that the chemical properties of lipids determine whether or not the lipids can trigger the aggregation of α-synuclein, thus affecting the balance between functional and aberrant behavior of the protein.

Studies of proteins' formation of amyloid fibrils have revealed that potentially cytotoxic oligomers frequently accumulate during fibril formation. An important question in the context of mechanistic studies of this process is whether or not oligomers are intermediates in the process of amyloid fibril formation, either as precursors of fibrils or as species involved in the fibril elongation process or instead if they are associated with an aggregation process that is distinct from that generating mature fibrils. Here we describe and characterize in detail two well-defined oligomeric species formed by the protein α-synuclein (αSN), whose aggregation is strongly implicated in the development of Parkinson's disease (PD). The two types of oligomers are both formed under conditions where amyloid fibril formation is observed but differ in molecular weight by an order of magnitude. Both possess a degree of β-sheet structure that is intermediate between that of the disordered monomer and the fully structured amyloid fibrils, and both have the capacity to permeabilize vesicles in vitro. The smaller oligomers, estimated to contain ∼30 monomers, are more numerous under the conditions used here than the larger ones, and small-angle X-ray scattering data suggest that they are ellipsoidal with a high degree of flexibility at the interface with solvent. This oligomer population is unable to elongate fibrils and indeed results in an inhibition of the kinetics of amyloid formation in a concentration-dependent manner.

In nature, sophisticated functional materials are created through hierarchical self-assembly of simple nanoscale motifs. In the laboratory, much progress has been made in the controlled assembly of molecules into one-, two- and three-dimensional artificial nanostructures, but bridging from the nanoscale to the macroscale to create useful macroscopic materials remains a challenge. Here we show a scalable self-assembly approach to making free-standing films from amyloid protein fibrils. The films were well ordered and highly rigid, with a Young's modulus of up to 5-7 GPa, which is comparable to the highest values for proteinaceous materials found in nature. We show that the self-organizing protein scaffolds can align otherwise unstructured components (such as fluorophores) within the macroscopic films. Multiscale self-assembly that relies on highly specific biomolecular interactions is an attractive path for realizing new multifunctional materials built from the bottom up.

Parkinson’s disease is a highly debilitating neurodegenerative condition whose pathological hallmark is the presence in nerve cells of proteinacious deposits, known as Lewy bodies, composed primarily of amyloid fibrils of α-synuclein. Several missense mutations in the gene encoding α-synuclein have been associated with familial variants of Parkinson’s disease and have been shown to affect the kinetics of the aggregation of the protein. Using a combination of experimental and theoretical approaches, we present a systematic in vitro study of the influence of disease-associated single-point mutations on the individual processes involved in α-synuclein aggregation into amyloid fibrils. We find that lipid-induced fibril production and surface catalyzed fibril amplification are the processes most strongly affected by these mutations and show that familial mutations can induce dramatic changes in the crucial processes thought to be associated with the initiation and spreading of the aggregation of α-synuclein.

The self-assembly of molecular building blocks into nano-and micro-scale supramolecular architectures has opened up new frontiers in polymer science. Such supramolecular species not only possess a rich set of dynamic features as a consequence of the non-covalent nature of their core interactions, but also afford unique structural characteristics. Although much is now known about the manner in which such structures adopt their morphologies and size distributions in response to external stimuli, the kinetic and thermodynamic driving forces that lead to their transformation from soluble monomeric species into ordered supramolecular entities have remained elusive. Here we focus on Boc-diphenylalanine, an archetypical example of a peptide with a high propensity towards supramolecular self-organization, and describe the pathway through which it forms a range of nano-assemblies with different structural characteristics. Our results reveal that the nucleation process is multi-step in nature and proceeds by Ostwald's step rule through which coalescence of soluble monomers leads to the formation of nanospheres, which then undergo ripening and structural conversions to form the final supramolecular assemblies. We characterize the structures and thermodynamics of the different phases involved in this process and reveal the intricate nature of the transitions that can occur between discrete structural states of this class of supramolecular polymers.

Abstract. Parkinson's disease (PD) is characterized by proteinaceous aggregates named Lewy Bodies and Lewy Neurites containing α-synuclein fibrils. The underlying aggregation mechanism of this protein is dominated by a secondary process at mildly acidic pH, as in endosomes and other organelles. This effect manifests as a strong acceleration of the aggregation in the presence of seeds and a weak dependence of the aggregation rate on monomer concentration. The molecular mechanism underlying this process could be nucleation of monomers on fibril surfaces or fibril fragmentation. Here, we aim to distinguish between these mechanisms. The nature of the secondary processes was investigated using differential sedimentation analysis, trap and seed experiments, quartz crystal microbalance experiments and super-resolution microscopy. The results identify secondary nucleation of monomers on the fibril surface as the dominant secondary process leading to rapid generation of new aggregates, while no significant contribution from fragmentation was found. The newly generated oligomeric species quickly elongate to further serve as templates for secondary nucleation and this may have important implications in the spreading of PD.