Intramolecular Charge Interactions as a Tool to Control the Coiled‐Coil‐to‐Amyloid Transformation

Abstract

Under the influence of a changed environment, amyloid-forming proteins partially unfold and assemble into insoluble beta-sheet rich fibrils. Molecular-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 concentration, pH value, and ionic strength. The design strictly follows the characteristic heptad repeat of the alpha-helical coiled-coil structural motif. In all peptides, three valine residues, known to prefer the beta-sheet conformation, have been incorporated at the solvent-exposed b, c, and f positions to make the system prone to amyloid formation. Additionally, pH-controllable intramolecular 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 circular dichroism spectroscopic analysis and thioflavin T fluorescence, and the resulting aggregates were further characterized by transmission electron microscopy. Whereas uninterrupted alpha-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, alpha-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 observed 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 intramolecular charge repulsions in the model system presented herein.

How Post‐Translational Modifications Influence Amyloid Formation: A Systematic Study of Phosphorylation and Glycosylation in Model Peptides

“…[35] The impact of electrostatics on peptide and protein folding and self-assembly has been studied extensively. [11,36,37] Electrostatic interactions involving phosphate, which has a formal charge of À2, are among the strongest interactions involving the standard amino acids and can thus easily perturb the native PP structure. Intramolecular Coulomb repulsions between glutamate (at position b) and phosphate (at position f) destabilize the a-helical coiled coil and as a consequence promote unfolding (Figure 2b-d).…”

“…The introduction of a single bgalactose does not change the folding behavior of the model peptide, but does alter the aggregation kinetics in a site-specific manner. The presence of multiple galactose residues has an effect similar to that of phosphoryl- have directed their efforts towards developing simplified models for the studying of aggregation in response to diverse stimuli, including pH, [11,12] temperature, [13] metal ions, [14] and hydrophobic defects. [15] However, to our knowledge, only a few reports describe the influence of phospho-A C H T U N G T R E N N U N G rylA C H T U N G T R E N N U N G ation and glycosylation by using the same model system.…”

“…[11,19,20] We have explored the a-helical coiled coil folding motif, which is very common in nature and has been extensively studied in recent years, proving its application as a reliable model system. [9,11,19,20,41] Furthermore, the design principles of coiled coils are very well understood, [21] and folding is based on oligomerization, which is also the foundation of amyloid formation. Coiled coils consist of at least two a-helices wrapped around each other in a slight superhelical twist.…”

Energy landscapes of a mechanical prion and their implications for the molecular mechanism of long-term memory

“…This observation suggests that an external driving force will be needed in vivo. The α-helix to β-sheet (α-to-β) transition in other coiled coils has been shown to be enhanced by mechanical force (21,22), high temperature (23), pH change (24), and high protein concentration (25). A mechanical force-induced α-to-β transition has been observed in fibrin (22), collagen, and vimentin (26).…”

Selbstorganisation von Coiled‐Coils in der synthetischen Biologie: Inspiration und Fortschritt

“…Durch Verändern des pH-Wertes werden diese Reste geladen, wodurch das Coiled-Coil destabilisiert wird und sich in ein Zufallsknäuel oder eine b-Faltblattstruktur umlagert. [101] Ein reversiblerer Typ von Sekundärstrukturschalter beruht auf der Umschaltung von Coiled-Coils zu Zinkfingermotiven. Durch Einbringen von Motiven beider Faltungsformen in eine einzelne Sequenz falten stabile Coiled-Coils bei der Bindung eines Metalls in die stärker globuläre Zinkfingerkonformation.…”