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.

Background: Protein oligomers are implicated as cytotoxic membrane-disrupting agents in neurodegenerative diseases. Results: The small molecule EGCG, which inhibits ␣-synuclein oligomer toxicity, moderately reduces membrane binding and immobilizing the oligomer C-terminal tail. Conclusion:The ␣-synuclein oligomer may disrupt membranes by vesicle destabilization rather than pore formation. Significance: Limited reduction of oligomer membrane affinity may be sufficient to prevent cytotoxicity.

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.

The cytotoxic complex formed between α-lactalbumin and oleic acid (OA) has inspired many studies on protein-fatty acid complexes, but structural insight remains sparse. After having used small-angle X-ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein-oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core-shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off-loading of the OA into cell membranes.

The macro- and micro-features of bone can be assessed by using imaging methods. However, nano- and molecular features require more detailed characterization, such as use of e.g., vibrational spectroscopy and X-ray scattering. Nano- and molecular features also affect the mechanical competence of bone tissue. The aim of the present study was to reveal the effects of mineralization and its alterations on the mineral crystal scale, by investigating the spatial variation of molecular composition and mineral crystal structure across the cross-section of femur diaphyses in young rats, and healthy and osteoporotic mature rats (N=5). Fourier transform infrared spectroscopy and scanning small- and wide-angle X-ray scattering (SAXS/WAXS) techniques with high spatial resolution were used at identical locations over the whole cross-section. This allowed quantification of point-by-point information about the spatial distribution of mineral crystal volume. All measured parameters (crystal dimensions, degree of orientation and predominant orientation) varied across the cortex. Specifically, the crystal dimensions were lower in the central cortex than in the endosteal and periosteal regions. Mineral crystal orientation followed the cortical circumference in the periosteal and endosteal regions, but was less well-oriented in the central regions. Central cortex is formed rapidly during development through endochondral ossification. Since rats possess no osteonal remodeling, this bone remains (until old age). Significant linear correlations were observed between the dimensional and organizational parameters, e.g., between crystal length and degree of orientation (R(2)=0.83, p<0.001). Application of SAXS/WAXS provides valuable information on bone nanostructure and its constituents, effects of diseases and, prospectively, mechanical competence.