Phospholipid Ether Linkages Significantly Modulate the Membrane Affinity of the Antimicrobial Peptide Novicidin.

The biological activity of antimicrobial peptides is believed to be closely linked to their ability to perturb bacterial membranes. This makes it important to understand the basis of their membrane-binding properties. Here, we present a biophysical analysis of the interactions of the antimicrobial peptide Novicidin (Nc) with ether- and ester-linked C14 phospholipid vesicles below and above the lipid phase transition temperature (t p). These interactions are strongly dependent on whether the lipids contain ether or ester linkages. Nc is in random coil state in solution but undergoes a large increase in α-helicity in ether vesicles, and to a much smaller extent in ester vesicles, around the t p. This structure is lost at higher temperatures. Steady-state fluorescence and stopped-flow kinetics using fluorophore-labeled Nc reveal that Nc binds more strongly to ether vesicles than to ester vesicles below the t p, while there is no significant difference above the t p. This may reflect ether lipid interdigitation in the gel phase. Isothermal titration calorimetry reveals that partitioning of Nc into both lipids is exothermic and thus enthalpy driven. The higher enthalpy associated with binding to ether lipid may be linked to Nc's higher propensity to form α-helical structure in this lipid. The large effect of the ether-ester interchange reveals that membrane-AMP interactions can be strongly modulated by charge-neutral head group changes.

High stability and cooperative unfolding of α-synuclein oligomers.

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.

Formation of dynamic soluble surfactant-induced amyloid ß peptide aggregation intermediates.

Intermediate amyloidogenic states along the amyloid ß peptide (Aß) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aß aggregation, we here investigate surfactant-induced Aß aggregation. This process leads to co-aggregates featuring a ß-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aß in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via ß-structure to the fully formed α-helical state at high surfactant concentration. The ß-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aß co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k(ex) ∼1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with ß-structure promoting substances, such as surfactants, Aß is kinetically driven toward an aggregation-prone state.

The role of stable α-synuclein oligomers in the molecular events underlying amyloid formation.

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 ß-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.

Impact of UHT treatment and storage on liquid infant formula: Complex structural changes uncovered by centrifugal field-flow fractionation with multi-angle light scattering.

Protein modifications in liquid infant formula (IF) have been widely studied, but distinguishing between heat- and storage-induced structural changes remains challenging. A generic liquid IF was subjected to direct or indirect UHT treatment and stored at 40 °C up to 180 days. Colour and pH were monitored and structural changes were characterised by dynamic light scattering, SDS-PAGE and centrifugal field-flow fractionation (FFF) coupled with multi-angle light scattering (MALS) and UV detectors to evaluate whether heat-induced differences would level out during storage. Both direct- and indirect UHT treatment led to structural changes, where the higher heat load of the indirect UHT treatment caused more pronounced changes. Indications were that storage-induced changes in pH, browning and non-reducible cross-links were not dependent on UHT treatment. However, FFF-MALS-UV analysis allowed characterisation of complex aggregates, where structural changes continued to be most pronounced in indirect UHT treated samples, and different storage-induced aggregation behaviour was observed.

The N-terminus of α-synuclein is essential for both monomeric and oligomeric interactions with membranes.

The intrinsically disordered protein α-synuclein (αSN) is linked to Parkinson's Disease and forms both oligomeric species and amyloid fibrils. The N-terminal part of monomeric αSN interacts strongly with membranes and αSN cytotoxicity has been attributed to oligomers' ability to interact with and perturb membranes. We show that membrane folding of monomeric wt αSN and N-terminally truncated variants correlates with membrane permeabilization. Further, the first 11 N-terminal residues are crucial for monomers' and oligomers' interactions with and permeabilization of membranes. We attribute oligomer permeabilization both to cooperative electrostatic interactions through the N-terminus and interactions mediated by hydrophobic regions in the oligomer.

Wildtype and A30P mutant alpha-synuclein form different fibril structures.

Parkinson's Disease (PD) is a neurodegenerative movement disorder affecting millions of people worldwide. One of the key players in the development of the disease is the protein α-synuclein (aSN), which aggregates in the brain of PD patients. The aSN mutant A30P has been reported to cause early-onset familial PD and shows different aggregation behavior compared to wt aSN. Here we use a multidisciplinary approach to compare the aggregation process of wt and A30P aSN. In agreement with previous studies, we observe an initial lag phase followed by a continuous structural development of fibrils until reaching an apparent monomer-aggregate equilibrium state and a plateau in Thioflavin T (ThT) fluorescence intensity. However, at later timepoints A30P shows greater propensity than αSN wt to form dense bundled fibril networks. Combining small angle x-ray scattering, x-ray fibre diffraction and linear dichroism, we demonstrate that while the microscopic structure of the individual fibril essentially remains constant throughout the experiment, the formation of dense A30P fibril networks occur through a continuous assembly pathway while the formation of less dense wt fibril networks with fewer contact points follows a continuous path during the elongation phase and a second rearrangement phase after reaching the ThT fluorescence plateau. Our work thus highlights that structural rearrangements proceed beyond the plateau in ThT-based monitoring of the fibrillation process, and the density and morphology of the resulting fibril networks is highly dependent on the aSN form studied.

Multiple roles of heparin in the aggregation of p25α.

The 219-residue protein p25α stimulates the fibrillation of α-synuclein (αSN) in vitro and colocalizes with it in several α-synucleinopathies. Although p25α does not fibrillate by itself under native conditions in vitro, αSN-free p25α aggregates have also been observed in vivo in, for example, multiple system atrophy. To investigate which environmental conditions might trigger this aggregation, we investigated the effect of polyanionic biomolecules on p25α aggregation. Heparin, polyglutamate, arachidonic acid micelles, and RNA all induce p25α aggregation. More detailed studies using heparin as template for aggregation reveal that a minimum of 10-14 heparin monosaccharide units per heparin polymer are required. Bona fide fibrils are only formed at intermediate heparin concentrations, possibly because an excess of heparin binding sites blocks the inter-p25α contacts required for amyloid formation. Other polyanions also show an optimum for amyloid formation. Aggregation involves only modest structural changes according to both spectroscopic and proteolytic experiments. The aggregates do not seed aggregation of heparin-free p25α, suggesting that heparin is required in stoichiometric amounts to form organized structures. We are able to reproduce these observations in a model involving two levels of binding of p25α to heparin. We conclude that the modest structural changes that p25α undergoes can promote weak intermolecular contacts and that polyanions such as heparin play a central role in stabilizing these aggregates but in multiple ways, leading to different types of aggregates. This highlights the role of non-protein components in promoting protein aggregation in vivo.