The effect of post translational modification and oligomerisation on the structure-function relationship of horseradish and fluorescent proteins

Protein function is inherently linked to amino acid sequence; however, this vocabulary is innately limited. One natural method to supplement protein chemistry is the application of PTM. This modification has significant implications for both the structure and function of protein and can now be emulated with non-natural chemistry. This thesis examines the influence PTM has, on protein stability (Chapter 3), function (Chapter 4), structure (Chapter 5), and how nnPTM can be applied to induce functional communication (Chapters 6 and 7). This thesis first explores the expression of HRP in E. coli (Chapter 3), wherein it is demonstrated that soluble HRP expression, is achieved by the inclusion of the full-length proto-HRP sequence. These proto-HRP regions are then shown to cleave, and additionally, it is also shown that the removal of unoccupied N-linked glycosylation sites from the surface of HRP decreases the rate of precipitation observed in recombinant HRP. Next, recombinant apo-HRP is investigated as a corrective agent in a commercial immunoassay (Chapter 4), to establish if the corrective function of glycosylated apo-HRP can be achieved without glycosylation. Evidence generated indicates that glycosylation of apo-HRP is essential for the removal of false-positive rogue signalling. Subsequently, Chapter 5 explores the influence of PTM on HRP's structural rigidity. Using fluorescence emission to assess the red edge excitation shift of W117, it is observed that both haem binding and glycosylation increase protein structural rigidity, and this is confirmed by CD spectra. The latter two chapters of this thesis explore the potential of chromophore communication which can be induced by nnPTM. Together, both in silico interface modelling, and nnAA incorporation, is utilised for the formation of two fluorescent dimers. Firstly, this system was applied to the structurally similar combination of sfGFP and mCherry (Chapter 6). In which the enhanced chromophore proximity resulted in a significant decrease in function of mCherry, contrary to expectation. In Chapter 7 sfGFP and cytochrome b562 were dimerised to generate dimeric protein which could transfer energy by way of FRET (Chapter 7). Lastly, both nnPTM and natural PTM were combined in the formation of trimeric protein which was linked by SPAAC and disulphide linkage.