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.

Uncontrolled misfolding of proteins leading to the formation of amyloid deposits is associated with more than 40 types of diseases, such as neurodegenerative diseases and type-2 diabetes. These irreversible amyloid fibrils typically assemble in distinct stages. Transitions among the various intermediate stages are the subject of many studies but are not yet fully elucidated. Here, we combine high-resolution atomic force microscopy and quantitative nanomechanical mapping to determine the self-assembled structures of the decapeptide hIAPP [20][21][22][23][24][25][26][27][28][29] , which is considered to be the fibrillating core fragment of the human islet amyloid polypeptide (hIAPP) involved in type-2 diabetes. We successfully follow the evolution of hIAPP 20-29 nanostructures over time, calculate the average thickening speed of small ribbon-like structures, and provide evidence of the coexistence of ribbon and helical fibrils, highlighting a key step within the self-assembly model. In addition, the mutations of individual side chains of wide-type hIAPP 20-29 shift this balance and destabilize the helical fibrils sufficiently relative to the twisted ribbons to lead to their complete elimination. We combine atomic force microscopy structures, mechanical properties, and solid-state NMR structural information to build a molecular model containing β sheets in cross-β motifs as the basis of selfassembled amyloids.μFS | mutants | nanomechanical map | self-assembly nanostructure P rotein aggregation and amyloid deposits (1, 2) are associated with more than 40 different diseases (3) ranging from neurodegenerative diseases such as Alzheimer's disease (4) and Parkinson disease (5) to systemic amyloidosis, such as type-2 diabetes mellitus (T2D) (6). Over the last few decades, amyloid structures and amyloid assembly have been extensively studied using a variety of diffraction techniques such as X-ray scattering and electron diffraction. However, these methods only provide average structures (7). Many structures have also been resolved in great detail by solid-state NMR spectroscopy (8, 9), which mainly represents end-point structures and, unless specifically trapping intermediates, typically does not provide a clear picture of the transient structures formed during the fibrillation process. However, it is very important to resolve the dynamics of the nanostructures of amyloids at various steps during self-assembly to understand the mechanics behind amyloid initialization, formation, growth, and maturation as well as for the design of potential drugs. Atomic force microscopy (AFM) is capable of obtaining nanoscale resolution of individual molecules or supermolecular structures. This method also allows for the analysis of the selfassembly mechanism and the driving force of aggregation (10-14). Importantly, AFM, furthermore, provides the possibility to follow the dynamics to obtain a detailed picture of the amyloid assembly process (15).In the case of T2D, amyloid deposits, composed mainly of human islet amyloid polypeptide (hIA...

The deposition of amyloid material has been associated with many different diseases. Although these diseases are very diverse the amyloid material share many common features such as cross-β-sheet structure of the backbone of the proteins deposited. Another common feature of the aggregation process for a wide variety of proteins is the presence of prefibrillar oligomers. These oligomers are linked to the cytotoxicity occurring during the aggregation of proteins. These prefibrillar oligomers interact extensively with lipid membranes and in some cases leads to destabilization of lipid membranes. This interaction is however highly dependent on the nature of both the oligomer and the lipids. Anionic lipids are often required for interaction with the lipid membrane while increased exposure of hydrophobic patches from highly dynamic protein oligomers are structural determinants of cytotoxicity of the oligomers. To explore the oligomer lipid interaction in detail the interaction between oligomers of α-synuclein and the 4th fasciclin-1 domain of TGFBIp with lipid membranes will be examined here. For both proteins the dynamic species are the ones causing membrane destabilization and the membrane interaction is primarily seen when the lipid membranes contain anionic lipids. Hence the dynamic nature of oligomers with exposed hydrophobic patches alongside the presence of anionic lipids could be essential for the cytotoxicity observed for prefibrillar oligomers in general. This article is part of a Special Issue entitled: Lipid-protein interactions.

Many neurodegenerative diseases are linked with formation of amyloid aggregates. It is increasingly accepted that not the fibrils but rather oligomeric species are responsible for degeneration of neuronal cells. Strong evidence suggests that in Parkinson's disease (PD), cytotoxic α-synuclein (αSN) oligomers are key to pathogenicity. Nevertheless, insight into the oligomers' molecular properties remains scarce. Here we show that αSN oligomers, despite a large amount of disordered structure, are remarkably stable against extreme pH, temperature, and even molar amounts of chemical denaturants, though they undergo cooperative unfolding at higher denaturant concentrations. Mutants found in familial PD lead to slightly larger oligomers whose stabilities are very similar to that of wild-type αSN. Isolated oligomers do not revert to monomers but predominantly form larger aggregates consisting of stacked oligomers, suggesting that they are off-pathway relative to the process of fibril formation. We also demonstrate that 4-(dicyanovinyl)julolidine (DCVJ) can be used as a specific probe for detection of αSN oligomers. The high stability of the αSN oligomer indicates that therapeutic strategies should aim to prevent the formation of or passivate rather than dissociate this cytotoxic species.