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Proteinaceous deposits of α-synuclein amyloid fibrils are a hallmark of human disorders including Parkinson's disease. The onset of this disease is also associated with five familial mutations of the gene encoding the protein. However, the mechanistic link between single point mutations and the kinetics of aggregation, biophysical properties of the resulting amyloid fibrils, and an increased risk of disease is still elusive. Here, we demonstrate that the disease-associated mutations of α-synuclein generate different amyloid fibril polymorphs compared to the wild type protein. Remarkably, the α-synuclein variants forming amyloid fibrils of a comparable structure, morphology, and heterogeneity show similar microscopic steps defining the aggregation kinetics. These results demonstrate that a single point mutation can significantly alter the distribution of fibrillar polymorphs in α-synuclein, suggesting that differences in the clinical phenotypes of familial Parkinson's disease could be associated with differences in the mechanism of formation and the structural characteristics of the aggregates.

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Proteins fold into a single structural ensemble but can also misfold into many diverse structures including small aggregates and fibrils, which differ in their toxicity. The aggregate surface properties play an important role in how they interact with the plasma membrane and cellular organelles, potentially inducing cellular toxicity, however, these properties have not been measured to date due to the lack of suitable methods. Here, we used a spectrally resolved, super-resolution imaging method combined with an environmentally sensitive fluorescent dye to measure the surface hydrophobicity of individual aggregates formed by the protein α-synuclein (αS), whose aggregation is associated with Parkinson's disease. We show that the surface of soluble oligomers is more hydrophobic than fibrils and populates a diverse range of coexisting states. Overall, our data show that the conversion of oligomers to fibril-like aggregates and ultimately to fibrils results in a reduction in both hydrophobicity and the variation in hydrophobicity. This funneling characteristic of the energy landscape explains many of the observed properties of αS aggregates and may be a common feature of aggregating proteins.

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Competing models exist in the literature for the relationship between mutant Huntingtin exon 1 (Httex1) inclusion formation and toxicity. In one, inclusions are adaptive by sequestering the proteotoxicity of soluble Httex1. In the other, inclusions compromise cellular activity as a result of proteome co-aggregation. Using a biosensor of Httex1 conformation in mammalian cell models, we discovered a mechanism that reconciles these competing models. Newly formed inclusions were composed of disordered Httex1 and ribonucleoproteins. As inclusions matured, Httex1 reconfigured into amyloid, and other glutamine-rich and prion domain-containing proteins were recruited. Soluble Httex1 caused a hyperpolarized mitochondrial membrane potential, increased reactive oxygen species, and promoted apoptosis. Inclusion formation triggered a collapsed mitochondrial potential, cellular quiescence, and deactivated apoptosis. We propose a revised model where sequestration of soluble Httex1 inclusions can remove the trigger for apoptosis but also co-aggregate other proteins, which curtails cellular metabolism and leads to a slow death by necrosis.

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Super-resolution microscopy allows biological systems to be studied at the nanoscale, but has been restricted to providing only positional information. Here, we show that it is possible to perform multi-dimensional super-resolution imaging to determine both the position and the environmental properties of single-molecule fluorescent emitters. The method presented here exploits the solvatochromic and fluorogenic properties of nile red to extract both the emission spectrum and the position of each dye molecule simultaneously enabling mapping of the hydrophobicity of biological structures. We validated this by studying synthetic lipid vesicles of known composition. We then applied both to super-resolve the hydrophobicity of amyloid aggregates implicated in neurodegenerative diseases, and the hydrophobic changes in mammalian cell membranes. Our technique is easily implemented by inserting a transmission diffraction grating into the optical path of a localization-based super-resolution microscope, enabling all the information to be extracted simultaneously from a single image plane.

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Networks of nanoscale fibrous coatings made from self-assembled peptides are promising candidates for biomaterials that can promote the growth of mammalian cells. One particularly attractive feature is the possibility of adding biofunctional sequences to peptides to promote cell attachment. We deconvolute the topographic and chemical effects of nanoscale fibrils on cells by depositing a plasma polymer film on TTR1-based fibrils decorated with a range of cell adhesive chemistries (RGD and cycloRGDfK), producing a surface that retains the nanoscale fibrous topography of surface-bound fibrils but lacks the fibril surface chemistry. The surface topography was found to influence cell toxicity and spreading, and the fibril surface chemistry influenced cell attachment and spreading. This study highlights the importance of considering both the chemical and physical features of novel biomaterials and illustrates the use of plasma polymer deposition as a tool for examining the relationship between amyloid fibril structure and function.

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A growing number of protein-based fibrous biomaterials have been produced with a cross-ß amyloid core yet the long-term effect of these materials on cell viability and the influence of core and non-core protein sequences on viability is not well understood. Here, synthetic bioactive TTR1-RGD and control TTR1-RAD or TTR1 fibrils were used to test the response of mammalian cells. At high fibril concentrations cell viability was reduced, as assessed by mitochondrial reduction assays, lactate dehydrogenase membrane integrity assays and apoptotic biomarkers. This reduction occurred despite the high density of RGD cell adhesion ligands and use of cells displaying integrin receptors. Cell viability was affected by fibril size, maturity and whether fibrils were added to the cell media or as a pre-coated surface layer. These findings show that while cells initially interact well with synthetic fibrils, cellular integrity can be compromised over longer periods of time, suggesting a better understanding of the role of core and non-core residues in determining cellular interactions is required before TTR1-based fibrils are used as biomaterials.

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Amyloid fibrils are ordered, insoluble protein aggregates that are associated with neurodegenerative conditions such as Alzheimer's disease. The fibrils have a common rod-like core structure, formed from an elongated stack of ß-strands, and have a rigidity similar to that of silk (Young's modulus of 0.2-14 GPa). They also exhibit high thermal and chemical stability and can be assembled in vitro from short synthetic non-disease-related peptides. As a result, they are of significant interest in the development of self-assembled materials for bionanotechnology applications. Synthetic DNA molecules have previously been used to form intricate structures and organize other materials such as metal nanoparticles and could in principle be used to nucleate and organize amyloid fibrils. Here, we show that DNA origami nanotubes can sheathe amyloid fibrils formed within them. The fibrils are built by modifying the synthetic peptide fragment corresponding to residues 105-115 of the amyloidogenic protein transthyretin and a DNA origami construct is used to form 20-helix DNA nanotubes with sufficient space for the fibrils inside. Once formed, the fibril-filled nanotubes can be organized onto predefined two-dimensional platforms via DNA-DNA hybridization interactions.

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Mutations in the polypeptide sequence that forms the core structure of amyloid fibrils are known to impact on fibril assembly and stability but the effect of changes on noncore residues, particularly relating to functionalized fibrils where the fibril core is preserved, has not been systematically examined. In this study, the short peptide sequence TTR(105-115) (also known as TTR1) and the functionalized variants TTR1-RGD and TTR1-RAD are used as a model system to investigate the effect of noncore residues on the kinetics of fibril assembly. The noncore residues in TTR1-RGD and TTR1-RAD influence the rate of fibril assembly in non-seeded samples with the glycine residue at position 15 increasing the rate of aggregation compared to alanine. Mature TTR1-RGD fibrils were also found to fragment more readily, indicating possible differences in mechanical properties. Fragments of each type of fibril are capable of self- and cross-seeding, generating fibrils with a highly similar cross-ß core structure. The similar rates of assembly observed for self-seeded samples reflect the similar free energy of elongation calculated for these peptides, while the morphology of cross-seeded fibrils is determined by the properties of the monomeric peptide and its macromolecular arrangement within the protofilaments and fibrils. These findings illustrate that noncore residues impact on fibril formation and fibril properties and demonstrate that the influence of noncore residues should be considered when designing sequences for the production of self-assembling functional fibrillar materials.

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Amino acid-based core cross-linked star (CCS) polymers (poly(L-lysine)(arm)poly(L-cystine)(core)) with peripheral allyl functionalities were synthesized by sequential ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides (NCAs) via the arm-first approach, using N-(trimethylsilyl)allylamine as the initiator. Subsequent functionalization with a poly(ethylene glycol) (PEG)-folic acid conjugate via thiol-ene click chemistry afforded poly(PEG-b-L-lysine)(arm)poly(L-cystine)(core) stars with outer PEG coronas decorated with folic acid targeting moieties. Similarly, a control was prepared without folic acid, using just PEG. A fluorophore was used to track both star polymers incubated with breast cancer cells (MDA-MB-231) in vitro. Confocal microscopy and flow cytometry revealed that the stars could be internalized into the cells, and higher cell internalization was observed when folic acid moieties were present. Cytotoxicity studies indicate that both stars are nontoxic to MDA-MB-231 cells at concentrations of up to 50 µg/mL. These results make this amino acid-based star polymer an attractive candidate in targeted drug delivery applications including chemotherapy.

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Peptide self-assembly offers a route for the production of fibrous nanomaterials with advanced bioactive properties that promote specific cell interactions. In this study the peptide TTR1-cycloRGDfK was designed to form amyloid-like fibrils that display the functional cyclic RGDfK pentapeptide ligand to target mammalian cell surface α(V)ß3 integrin receptors. The TTR105₋115 (or TTR1) sequence was used as the self-assembling domain. Once assembled, TTR1-cycloRGDfK fibrils display a characteristic cross-ß core structure by X-ray fibre diffraction that was preserved following dehydration. Thin films of fibrils were characterised by infrared synchrotron mapping, scanning electron microscopy and atomic force microscopy. Cell adhesion and spreading were promoted on thin films of TTR1-cycloRGDfK fibrils via specific interactions with the cyclic RGDfK ligand. Low levels of non-specific interactions were also observed between cells and non-functionalised fibrils. TTR1-cycloRGDfK fibrils are an advance on bioactive fibrils previously designed to interact with a range of RGD binding integrins and our findings show that the assembly of amyloid-like fibrils based on the TTR1 sequence is robust and can be directed to form materials with specific properties.

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