The interaction of ß-amyloid model peptides with lipid membranes

The aim of this work was to characterize the structural modifications of model beta-amyloid peptides and the lipid membrane upon binding. The first step of this thesis was to reproduce the random-coil to beta-sheet structural transition of bAP(25-35). It appeared that additional energy, brought by stirring or heating, acts as a catalyst of the structural transition. Also the presence of inhomogeneous aggregates of bAP(25-35) in the solution influences the CD spectra and thereby the analysis of the peptide secondary structure. This work points out the experimental factors that can strongly influence bAP(25-35) secondary structure and make biophysical studies difficult. In this study, an alpha-helical intermediate was observed for the first time in the random-coil to beta-sheet transition of bAP(25-35).
bAP(25-35) has no chromophore and the quantification of the peptide content was difficult, in particular under conditions of aggregation. Amyloid model peptides were therefore synthesized. The insertion of Trp either at position 24 or 32 of bAP(25-35) resulted in two analog peptides, WbAP(25-35) and bAP(25-35)_I32W. They form aggregates in aqueous buffers at micromolar peptide concentrations, similar to bAP(25-35). HFIP pretreatment homogenizes WbAP(25-35) solutions at physiological and acidic pH values. The aggregates of bAP(25-35)_I32W are small and no effect of HFIP pretreatment was observed. Aggregates with sizes ranging from 60 to 600 nm are measured with dynamic light scattering for the three pretreated peptides. Nevertheless fibril formation of the two analogs could not be observed with Thioflavin T fluorescence, in contrast to bAP(25-35). The presence of Trp in WbAP(25-35) and bAP(25-35)_I32W seems to hinder the binding of the fluorescent dye. Our work opens new possibilities in the design of amyloid model peptides that can facilitate structural and thermodynamic studies.
CD spectroscopy showed that the presence and position of Trp influences the secondary structure of the free and the lipid-bound amyloid model peptides. The lipid membrane favors the random-coil structure of bAP(25-35) and WbAP(25-35) at low peptide concentration. This shows the dual role of the membrane which can enhance beta-sheet content in the peptide structure at higher peptide concentrations. Thermodynamic parameters of the peptide-to-lipid binding could be determined at pH 4.0. In addition, we demonstrated with solid-state NMR that the insertion of the amyloid model peptides into the lipid membrane influences the fluidity of the lipid acyl chains. In contrast, the association of aggregated amyloid model peptides from the aqueous phase with the lipid membrane leads to the formation of an isotropic phase. This phenomenon is reported here for the first time and might be correlated with the toxicity of the amyloid peptide on the neuronal cells.
The NMR study of DMPG at high lipid content proved that DMPG is in a bilayer state at low and high salt content. At high ionic strength, the phase transition occurs at 24°C. At low ionic strength, two distinct bilayer phases are observed on the NMR spectra; they are characterized by a broad phase transition between 20 and 24°C. The results obtained at low lipid content and high ionic strength are similar to those at high lipid content: bilayer features are observed on the whole temperature range and the gel-to-liquid-crystal transition occurs at 23°C, as proved with DSC and NMR data. At low lipid content and low ionic strength, bilayer characteristics are also observed on the whole temperature range in the 2H-NMR spectra. In contrast, during the phase transition, an isotropic line is observed in the 31P-NMR spectra. Spectral fitting indicated that at low ionic strength, the phase transition is characterized by enhanced motions of the acyl chains and a change in the orientation of the phosphoglycerol headgroup. In addition, we observed thixotropy in all samples upon prolonged heating, independently of the lipid or salt content.