Abstract:
Bionanotechnology is the utilisation of biological material to build nanotechnologies and nanomaterials. This field has seen many improvements in recent years, especially in the development of nanomaterials made of proteins. Proteins naturally cover a vast landscape of structures, functions, and complexities, making them ideal candidates for development as next generation nanomaterials. This thesis is concerned with modifying the protein human peroxiredoxin 3 (Prx) to increase its usability as a nanomaterial. Natively, Prx oligomerises into a range of useful shapes (dimers, rings, and tubes) that we can controllably switch between, is structurally stable, and is easy to make. These properties make Prx interesting for use as a biomaterial, but can yet be built upon. Much work has been conducted to increase the understanding over how Prx switches between oligomeric states, and in increasing the control we have over these oligomer switches. However, we still do not fully understand how the protein assembles into tubes. Within this thesis I use a mutation study to explore what chemical interactions take place to allow these assemblies to happen in different conditions, finding that the protein stacks through electrostatic interactions and is stabilised by a polar bond network. Further to this, I explore the effect a variety of metals have on Prx oligomerisation. Specifically, how transition metals stabilise high molecular weight structures, and how heavy metals can inhibit high molecular weight structure formation. The native function of Prx is as an antioxidant, however this function alone is not necessarily useful for a nanomaterial. Therefore, I have also worked on increasing the functionality of Prx. To do this, I incorporated the unnatural amino acid (unnatural amino acid) azidophenylalanine into the polypeptide chain of Prx, allowing the protein to go through site specific click reactions. This is the first time an unnatural amino acid has been incorporated into a peroxiredoxin protein. Although I found that azidophenylalanine can be reduced to an unusable aminophenylalanine, under non-reducing conditions we can attach moieties onto Prx to an 80 % efficiency. After further modifying Prx to have a stabilised ring as well as an unnatural amino acid, I have successfully attached both small molecules like fluorophores, and larger molecules including the proteins green fluorescent protein and glucose oxidase. As such, Prx has been made into a working nanomaterial in the form of a nanoscaffold.