The formation of amyloid aggregates is responsible for a wide range of diseases, including Alzheimer's and Parkinson's disease. Although the amyloid-forming proteins have different structures and sequences, all undergo a conformational change to form amyloid aggregates that have a characteristic cross-β-structure. The mechanistic details of this process are poorly understood, but different strategies for the development of inhibitors of amyloid formation have been proposed. In most cases, chemically diverse compounds bind to an elongated form of the protein in a β-strand conformation and thereby exert their therapeutic effect. However, this approach could favor the formation of prefibrillar oligomeric species, which are thought to be toxic. Herein, we report an alternative approach in which a helical coiled-coil-based inhibitor peptide has been designed to engage a coiled-coil-based amyloid-forming model peptide in a stable coiled-coil arrangement, thereby preventing rearrangement into a β-sheet conformation and the subsequent formation of amyloid-like fibrils. Moreover, we show that the helix-forming peptide is able to disassemble mature amyloid-like fibrils.

Self-assembling peptides can be used to create tunable higher-order structures for the multivalent presentation of a variety of ligands. We describe a novel, fiber-forming coiled-coil-based peptide that assembles to display, simultaneously, carbohydrate and peptide ligands recognized by biomacromolecules. Preassembly decoration of the scaffold with a diphtheria toxin peptide epitope or a mannose motif did not interfere with self-assembly of the nanostructure. The resulting multivalent display led to tighter binding by antidiphtheria toxin antibodies and mannose-specific carbohydrate binding proteins, respectively. The potential of this self-assembling peptide to display ligands in bioanalytical assays is illustrated by its decoration with a disaccharide glycotope from the Leishmania parasite. Carbohydrate-specific antibodies produced in response to a Leishmania infection are detected more sensitively in human and canine sera due to the multivalent presentation on the self-assembled scaffold. Thus, nanofibers based on coiled-coil peptides are a powerful tool for the development of bioassays and diagnostics.

The ability to adopt at least two different stable conformations is a common feature of proteins involved in many neurodegenerative diseases. The involved molecules undergo a conformational transition from native, mainly helical states to insoluble amyloid structures that have high β-sheet content. A detailed characterization of the molecular architecture of highly ordered amyloid structures, however, is still challenging. Their intrinsically low solubility and high tendency to aggregate often considerably limits the application of established high-resolution techniques such as NMR and X-ray crystallography. An alternative approach to elucidating the tertiary and quaternary organization within an amyloid fibril is the systematic replacement of residues with amino acids that exhibit special conformational characteristics, such as glycine and proline. Substitutions within the β-sheet-prone sequences of the molecules usually severely affect their ability to form fibrils, whereas incorporation at external loop- and bend-like positions often has only marginal effects. Here we present the characterization of the internal architecture of a de novo designed coiled-coil-based amyloid-forming model peptide by means of a series of systematic single glycine and proline replacements in combination with a set of simple low-resolution methods. The folding and assembly behavior of the substituted peptides was monitored simultaneously using circular dichroism spectroscopy, Thioflavin T fluorescence staining, and transmission electron microscopy. On the basis of the obtained data, we successfully identify characteristic bend and core positions within the peptide sequence and propose a detailed structural model of the internal fibrillar arrangement.

A weakly acidic pH-responsive polypeptide is believed to have the potential for an endosome escape function in a polypeptide-triggered delivery system. For constructing a membrane fusion device with pH-responsiveness, we have designed novel polypeptides that are capable of forming an α2 coiled coil structure. Circular dichroism spectroscopy reveals that a polypeptide, AP-LZ(EH5), with a Glu and His salt-bridge pair at a staggered position in the hydrophobic core forms a stable coiled coil structure only at endosomal pH values (pH 5.0 to 5.5). On the basis of their endosomal-pH responsiveness, a boronic acid/polypeptide conjugate (BA-H5-St) was also designed as a pilot molecule to construct a pH-responsive, one-way membrane fusion system with a sugarlike compound (phosphatidylinositol: PI)-containing liposome as a target. Membrane fusion behavior was characterized by lipid-mixing, inner-leaflet lipid-mixing, and contents-mixing assays. These studies reveal that membrane fusion is clearly observed when the pH of the experimental system is changed from 7.4 (physiological condition) to 5.0 (endosomal condition).

The binding interaction between aggregates of the 5-chloro-2-[[5-chloro-3-(3-sulfopropyl)-3H-benzothiazol-2-ylidene]methyl]-3-(3-sulfopropyl)benzothiazolium hydroxide inner salt ammonium salt (CD-1) and alpha-helix, as well as beta-sheet forming de novo designed peptides, was investigated by absorption spectroscopy, circular dichroism spectroscopy, and cryogenic transmission electron microscopy. Both pure dye and pure peptides self-assembled into well-defined supramolecular assemblies in acetate buffer at pH = 4. The dye formed sheetlike and tubular H- and J-aggregates and the peptides alpha-helical coiled-coil assemblies or beta-sheet rich fibrils. After mixing dye and peptide solutions, tubular aggregates with an unusual ultrastructure were found, most likely due to the decoration of dye tubes with monolayers of peptide assemblies based on the strong electrostatic attraction between the oppositely charged species. There was neither indication of a transfer of chirality from the peptides to the dye aggregates nor the opposite effect of a structural transfer from dye aggregates onto the peptides secondary structure.