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[摘要]：Under the influence of a changed environment, amyloid-forming proteins partially unfold and assemble into insol. b-sheet rich fibrils. Mol.-level characterization of these assembly processes has been proven to be very challenging, and for this reason several simplified model systems have been developed over recent years. Herein, we present a series of three de novo designed model peptides that adopt different conformations and aggregate morphologies depending on concn., pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the a-helical coiled-coil structural motif. In all peptides, three valine residues, known to prefer the b-sheet conformation, have been incorporated at the solvent-exposed b, c, and f positions to make the system prone to amyloid formation. Addnl., pH-controllable intramol. electrostatic repulsions between equally charged lysine (peptide A) or glutamate (peptide B) residues were introduced along one side of the helical cylinder. The conformational behavior was monitored by CD spectroscopic anal. and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted a-helical aggregates are found at neutral pH, Coulomb repulsions between lysine residues in peptide A destabilize the helical conformation at acidic pH values and trigger an assembly into amyloid-like fibrils. Peptide B features a glutamate-based switch functionality and exhibits opposite pH-dependent folding behavior. In this case, a-helical aggregates are found under acidic conditions, whereas amyloids are formed at neutral pH. To further validate the pH switch concept, peptide C was designed by including serine residues, thus resulting in an equal distribution of charged residues. Surprisingly, amyloid formation is obsd. at all pH values investigated for peptide C. The results of further investigations into the effect of different salts, however, strongly support the crucial role of intramol. charge repulsions in the model system presented herein. |
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