Engineering functional amyloid fibrils for biomaterial applications

© 2012 Dr. Marie N. BongiovanniThe natural ability of biological molecules to self-assemble provides a useful route for the production of nanomaterials with desirable properties. Amyloid fibrils are a class of self-assembling proteins that are defined by their common core structure, which is rich in β-sheet structure. Fibrils are typically ~10 nm in width and have an elongated morphology with a length ~1 µm. These fibrils are historically associated with disease but they are also the functional state of some proteins in nature and can be produced in a controlled way from non-disease proteins. The fibril core structure imparts excellent physical properties such as a flexibility similar to silk and strength similar to steel, which recommend fibrils for applications in materials science. There is growing interest in the production of fibrils with useful properties, although a number of challenges remain before these materials can be applied. This thesis employed the TTR105-115 peptide, also known as TTR1, to drive the assembly of functional peptides into amyloid fibrils. The TTR1 sequence was selected for two reasons. Firstly, this peptide has a high propensity to self-assemble into highly ordered fibrous structures, where the peptides are arranged in a cross-β core structure. The TTR1 peptide also has a proven ability to drive the assembly of functional groups, so that they are displayed away from the fibril core at the C-terminal end of the TTR105-115 based peptide. The influence of these groups on the properties of assembled fibrils was investigated and the role functional groups on the kinetics of fibril assembly were determined. The TTR1-cycloRGDfK peptide was also designed with the aim of producing a fibril that would display specific properties. The peptide incorporated the functional cyclic RGDfK pentapeptide ligand, which has a high affinity and specificity towards the mammalian cell surface αVβ3 integrin receptor. Fibrils assembled from the TTR1-cycloRGDfK peptide were shown to promote the attachment and spreading of adherent mammalian cells when fibrils were presented as a surface coated layer. These findings demonstrated that the selection of functional sequences is paramount to the properties of fibrils based on the TTR1 peptide. The kinetics of functional fibril assembly were characterised using an established set of TTR1-based peptides: TTR1, TTR1-RGD, TTR1-RAD and the novel TTR1-GGK peptide. The functional ligands that are excluded from the fibril core were found to influence both the lag time and elongation rate of fibril formation. The study of TTR1-GGK assembly was further extended to include a wide range of solution conditions including conditions of varying ionic strength, solution pH or solutions containing different salt ions. The addition of salt promoted fibril. The extent of this effect was dependent on the degree of charge shielding, ion selectivity and the Hofmeister effect. The structure of the mature fibril was largely unaltered when fibrils were assembled in the presence of salt ions, indicating that salts may be used to tune fibril formation. Overall, these measurements demonstrated that non-fibril core residues alter the propensity for fibril formation, even with differences of a single amino acid. The impact of non-fibril core groups on assembly should therefore be considered when designing sequences for the production of functional fibrils. Functional amyloid fibrils were also tested for their biocompatibility using cell viability assays and membrane integrity assays. Mature fibrils assembled from the peptides TTR1, TTR1-RGD and TTR1-RAD were the primary focus since these aggregates are the targets for applications in materials science. The overarching conclusions from this work are firstly that the TTR1 peptide is a robust system that can promote the assembly of functional fibrils and secondly that non-fibril core residues greatly influence select properties of assembled fibrils, while other core structural features of fibrils remain intact. Non-core residues determined the extent and specificity of fibril binding to the cell membrane. Layers of TTR1-cycloRGDfK fibrils promoted the attachment and spreading of cells, demonstrating that fibrils can be engineered to have a positive effect on cell processes. A greater understanding of fibril biocompatibility is needed, however, before functional fibrils may be applied to biotechnology applications where cell interactions may be involved