Structural modification of proteins and peptides for the creation of nanomaterials.

Abstract

Amyloid fibrils are highly ordered β-sheet structures that are formed from a variety of proteins in vivo, where they may have biological roles or, more commonly, are found associated with a broad range of diseases (known as amyloidoses). Due to the role of amyloid fibrils in disease and their potential as bionanomaterials, formation of amyloid and amyloid-like fibrils in vitro are exciting areas of research activity. This thesis explores the formation and characterisation of nanofibre structures, including amyloid and amyloid-like fibrils, from diverse peptides and proteins. The growing interest in protein nanofibres in the bionanotechnology industry has led to research into new nanofibre forming target peptides and proteins. In this thesis, Raphanus sativus antifungal peptide 19mer (RsAFP-19) and a mutant of β2-microglobulin where the 3rd arginine was replaced with an alanine (R3A β2-microglobulin), were examined for their amyloiogenicity and nanofibre-forming propensity. Generation of nanofibre structures was successful for both, and the characteristics of formation, fibril structure and morphology were explored via thioflavin t binding (ThT), transmission electron microscopy (TEM), atomic force microscopy (AFM) and X-ray fibre diffraction. Nanofibre formation was also further characterised for mammalian lens crystallin proteins. Their ability to form protein nanofibres from semi-pure and crude protein mixtures was established, and the resulting fibrils characterised, including for their amyloid-like characteristics. This work demonstrates the ability of crystallin proteins to form inexpensive base materials for use in the bionanotechnology industry. Inhibition of amyloid fibril formation is an area of current research activity for therapeutic purposes (against amyloidoses). α-Crystallin is a well-known molecular chaperone which traps intermediately structured target proteins, preventing them from both amorphous and ordered (amyloid fibril) aggregation. Like other crystallin proteins, α-crystallin can itself form amyloid fibrils under mildly denaturing conditions. The chaperone activity of αT- and αB-crystallin, in native, amorphously aggregated and fibrillar forms were assessed. Amyloid fibrils and amorphous aggregates derived from αT and αB-crystallin acted as chaperones, although with modified activity to their native (non-fibrillar) structures. αB-Crystallin fibrils displayed enhanced chaperone activity, compared to native αB-crystallin. The chaperone activity of αB-crystallin was also assessed after its immobilisation onto solid surfaces. Protein immobilisation of the chaperone αB-crystallin was achieved using plasma generated aldehyde polymerisation and Schiff-based covalent bonding of the proteins. Immobilisation was characterised using X-ray photoelectron spectroscopy, AFM and quartz crystal micrography. Immobilised αB-crystallin was shown to act 100-fold to 5000-fold more effectively as a chaperone than native solution αB-crystallin (dependent upon target protein and the type of stress it was exposed to). This research has established that αT- and αB-crystallin can retain chaperone activity, or may even show enhanced activity, under conditions of extreme structural perturbation. This thesis explores both the induction and inhibition of amyloid fibril, amyloidlike fibril and nanofibre formation, using a range of peptides and proteins, both pure and in crude mixtures. These protein nanofibre structures were characterised, to establish potential future use in bionanotechnology, kinetics and activity of amyloid and amyloid-like fibril formation and assessment of amyloid fibril inhibitors. This work further deliniated aspects of αT- and αB-crystallin chaperone ability, highlighting these proteins’ ability to act as effective chaperones under a broad range of conditions.