De novo designed proteins for applications in research and biotechnology

While the advances of the scientific community have enabled extraordinary improvements in the capabilities of synthetic biology, there is a continued desire in biotechnology for enhanced or entirely novel biological functions. As proteins are either directly or indirectly responsible for the vast majority of naturally occurring biological activities, the modification of peptide structures constitutes a promising approach to address the ambitions of biotechnology. Central to the work in this thesis is the recognition that naturally occurring protein structures are intangibly complex due to the relics of evolutionary processes, accumulated from years of blind natural selection. Chapter 1 introduces de novo protein design strategies that circumvent the use of naturally occurring peptide scaffolds, offering examples of tractable systems that have been generated to perform various biological functions, thus forming the justification for the experimental approach undertaken here. The experimental work detailed in chapters 3 and 4 aimed to develop a system that would enable the incorporation of carotenoids and acenes into the internal cavity of de novo-designed ‘maquette’ proteins by hydrophobic partitioning alone. The results of these sections demonstrated that the protein chassis had little or no effect in the photophysical properties of the incorporated chromophores, whilst providing enhanced stability and solubility in entirely aqueous solutions. Conversely, the experimental strategy outlined in chapter 5 aimed to introduce nuclei of high atomic mass into the maquette proteins in order to directly affect the photophysical properties of the bound chromophores through the spin-orbit coupling interaction. The results of the final experimental chapter demonstrated that de novo designed proteins could effectively interface with native biological systems and provide a mechanism to enable cofactor incorporation in vivo. Where appropriate, the results of each experimental section are discussed in relation to their impact on specific areas of research and potential applications in biotechnology.