The presence of ␤-sheets in the core of amyloid fibrils raised questions as to whether or not ␤-sheetcontaining proteins, such as transthyretin, are predisposed to form such fibrils. However, we show here that the molecular structure of amyloid fibrils differs more generally from the ␤-sheets in native proteins. This difference is evident from the amide I region of the infrared spectrum and relates to the distribution of the / dihedral angles within the Ramachandran plot, the average number of strands per sheet, and possibly, the ␤-sheet twist. These data imply that amyloid fibril formation from native ␤-sheet proteins can involve a substantial structural reorganization.

We report here a recombinant expression system that allows production of large quantities of Alzheimer's Ab(1-40) peptide. The material is competent to dissolve in water solutions with ''random-coil properties,'' although its conformation and factual oligomerization state are determined by the physico-chemical solution conditions. When dissolved in 50 mM sodium phosphate buffer (pH 7.4) at 37 C, the peptide is able to undergo a nucleated polymerization reaction. The aggregation profile is characteristically bipartite, consisting of lag and growth phase. From these curves we determined the lag time as well as the rate of aggregation. Both values were found to depend on peptide concentration and addition or formation of seeds. Moreover, they can vary considerably between apparently identical samples. These data imply that the nucleation event is under influence of a stochastic factor that can manifest itself in profound macroscopic differences in the aggregation kinetics of otherwise indistinguishable samples.

The mechanism of the horseradish peroxidase (HRP)-H 2 O 2 -catalysed polymerisation of aniline in the presence of AOT vesicles was investigated. AOT (= bis-(2-ethylhexyl)sulfosuccinate) served as vesicleforming surfactant and dopant for obtaining at pH = 4.3 and room temperature within 24 h under optimal reaction conditions the green emeraldine salt form of polyaniline in 90-95% yield. Based on UV/VIS/NIR and EPR measurements carried out during the polymerisation reaction, and based on changes in aniline and H 2 O 2 concentrations and HRP activity, a mechanism is proposed. According to this ''radical cation mechanism'' chain growth occurs on the vesicle surface through addition of aniline radical cations to the growing polymer chain. H 2 O 2 plays two essential roles, to oxidise the heme group of HRP, and to oxidise the growing polymer chain for allowing the stepwise addition of new aniline radical cations. The entire reaction can be divided into three kinetically distinct phases. In the first rapid phase (5-10 min), the actual polymer formation takes place to yield the emeraldine salt form of polyaniline in its bipolaron state. In the second and third slower phases (1-2 days) the bipolarons transform into polarons with unpaired electrons. During the reaction, the HRP activity is decreasing until the enzyme becomes inactive after polymer formation. Reactions carried out with partially deuterated anilines were analysed by 2 H magic-angle spinning (MAS) NMR spectroscopy to demonstrate the regioselectivity of the chain growth: para-coupling of the aniline units clearly dominates. Association of the formed polyaniline with the vesicle membrane is evident from cryo-TEM and SANS measurements.

The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection.

The formation of polypeptide aggregates represents a nucleated polymerization reaction in which an initial nucleation event (lag phase) is followed by the extension of newly formed nuclei into larger aggregates, including fibrils (growth phase). The efficiencies of these reactions relate to the lag time (lag phase) and to the rate of aggregation (growth phase), which can be determined from experimental aggregation curves. Here we present a mutagenic analysis in which we replace valine 18 of the Alzheimer's Ab(1-40) peptide with 17 different amino acids and determine its effect on the lag time, and therefore, on the propensity of nucleation. Comparison with various physico-chemical properties shows that nucleation is affected in a predictable manner depending on the b-sheet propensity and hydrophobicity of residue 18. In addition, we observe a direct proportionality between the lag time and the rate of aggregation. These data imply that the two reactions, nucleation and polymerization, are governed by very similar physicochemical principles and that they involve the formation of the same types of noncovalent interactions.