Atomic to mesoscopic structures of amyloids

Differences in aggregation pathways could lead to the formation of diverse amyloid nanostructures. Atomic-level structures include oligomers and 0D aggregates (nanoclusters, nanoparticles, nano triangles, squares, and loops), while mesoscopic structures include, 1D aggregates (protofibrils, nanofibrils, nanoribbons, and nanotubes), 2D aggregates (sheets, films, and membranes) and 3D amyloid plaques (amorphous aggregates observed in neurodegenerative diseases [70].

The study of amyloids over the past half-century led to the belief that amyloid fibrils and amorphous aggregates were the agents responsible for cytotoxicity observed in neurodegenerative diseases [71-73]. In sharp contrast, current growing evidence suggests that oligomers and 0D aggregates found in the early stage of aggregation pathway are responsible for the cytotoxicity rather than mature fibrils [74-76]. Oligomers are small molecular aggregates that later form fibrils. They are formed by assembling individual monomers (monomers from 2- 30) [77]. Indeed, oligomers are soluble in aqueous solutions and have different sizes and morphologies. They are usually known as spherical species due to EM observations. Oligomers are formed in early stages of aggregation pathway as a metastable species which in turn converts to energetically more favorable species. In addition to the

11 differences seen in size and shape compared to fibrils, oligomers do not contain cross-β structure which is specific to amyloid fibrils [78]. In the context of the secondary structure of oligomers, some of the oligomeric species are rich in β-sheets, while others contain random coil [79]. A recent study has shown that amyloid oligomers have the ability to self-replicate from the monomer at physiological temperatures and thereby promote protofilament nucleation and further assembly into fibrils [80]. Some of the amyloid oligomers exhibit crystalline morphology, for instance, peptide segments derived from Aβ and tau proteins [81, 82]. Apart from oligomers, other prefibrillar aggregates which are a nanoparticle, nanospheres, and annular oligomers are also believing to cause toxicity in neurodegenerative diseases. Prefibrillar aggregates or 0D nanostructures are aggregates or clusters without any prominent dimensional feature. A study conducted to identify sizes of toxic species involved in PrP aggregation has revealed that PrP nanoparticles with masses similar to 14-28 PrP molecules are the highest toxic initiator species found in PrP disease [73]. Furthermore, Guo et al have shown that triphenylalanine peptide self-assemble into nanospheres with significant β-sheet content [83]. Another study has demonstrated that lipid-induced nanosphere formation by Aβ(1-40) which could be a possible mechanism to occur in Alzheimer’s disease [84]. Some of the studies have suggested that annular oligomer formation is related to channel hypothesis which is one of the toxicity mechanisms found in neurodegenerative diseases. The Aβ protein or α-synuclein based annular oligomers insert themselves into the cell membrane and form ion channels which could lead to the calcium influx into cells [85].

The 1D amyloid nanostructures include protofilaments, protofibrils, and nanofibrils. These structures have high hierarchical morphology in which individual protofilaments self- assemble to form protofibrils which in turn packed into mature amyloid fibrils. Differences in packing of protofilaments give rise to a diverse range of morphologies including nanofibrils, nanotubes, and nanoribbons. Nanotubes are tube-like structures with well-defined morphology and hollow architecture. This bio-inspired group of nanostructures is widely implicated in material science and nanotechnology in the last decade. Nature provides a few examples of biological tubular assemblies based on peptides and proteins such as capsid of the tobacco mosaic virus, microtubules, some of the disease associated amyloid fibrils [12, 86, 87]. Lanreotide which is a somatostatin analog has been reported to form nanotubes with 2D hexagonal packing, the diameter of 244 Å and a wall thickness of 18 Å [88]. Further, they proposed that electrostatic interactions are an important factor in the formation of nanotubes in the hexagonal lattice. In addition, lanreotide has been used as a template for silica

12 nanotube fabrication [89]. Apart from single peptide or protein, nanotubes can also form by the closure of the helical ribbon [90]. Elsewhere, pH dependant nanotube formation was reported for triptorelin where conformational changes in the peptide lead to the formation of either small or large nanotubes depending on the pH [91]. Recently, Zhao et al has shown that nanotubes formed by symmetric amphiphilic peptide could be converted to nanofibrils by increasing acetonitrile concentration in their solvent system [92]. Interestingly, co-assembly of phenylalanine-based peptides of FF and Boc-FF (N-(tert-butoxycarbonyl)- L-Phe-L-Phe- COOH) has formed nanotubes. Variation in the molar ratio of these two peptides could give rise to nanotubes with different length distribution [93]. Nanotube diameter of lanreotide has been finely amended by chemically modifying the building blocks of the peptide [94]. Twisted ribbons and helical ribbons are the other frequently reported fibril morphologies. Twisted ribbons are identified by Gaussian curvature (saddle-like curvature), while helical ribbons are identified by zero Gaussian curvature. Twisted ribbon will convert to helical ribbon depending on a number of protofilaments associated which in turn closure to form nanotubes. Amyloidogenic hexapeptide ILQINS derived from hen egg-white lysozyme form right-handed helical ribbons and crystals [95]. The same group has shown that conversion of fibrils to the crystal in single aggregates occurs via untwisting of the twisted ribbon by using similar type peptide sequences as above [96].

Due to high mechanical properties and adherence properties, amyloids can be used in applications in material science and nanotechnology. Modifications to 1D structures could deliver a variety of 2D structures including, 2D amyloid films [97], membranes [98]and nanosheets [99].