This thesis examines the tolerance and effects of unnatural amino acid (Uaa) incorporation into proteins in Escherichia coli using an expanded genetic code. Uaa incorporation was used to alter or install new properties in the target proteins, superfolder Green Fluorescent Protein (sfGFP) and cytochrome b562. Chapter 3 deals with the technical aspects of Uaa incorporation including orthogonality of the machinery and yield. Substitution of residues in sfGFP for unnatural analogues of tyrosine was shown to be a valuable way of altering the properties of the protein. Variants were generated with red-shifted fluorescence and altered excitation spectra. The majority of this work focused on the Uaa, p-azido-L-phenylalanine (azPhe) as it has a number of properties that would be desirable for use in proteins such as photoreactivity and selective reactivity with alkynes. By incorporating azPhe into key residues of sfGFP, variants were created that could be controlled using light (Chapter 4). Light-dependant fluorescence activation, deactivation and switching were demonstrated in vitro and in live cells. The molecular basis for these changes was investigated by a combination of spectroscopy and X-ray crystallography in Chapter 5. The photoreactivity of azPhe was exploited for a different purpose in Chapter 6. Proteins were used as an alternative to synthetic cages for studying low temperature phenyl azide photochemistry. Here, two radicals (anilino and triplet phenyl nitrene) were successfully caged and detected on photolysis, with the radical observed dependant on the protein environment. Finally, in Chapter 7 the selective reactivity of azPhe was used to create proteins capable of site-specific modification (via Click chemistry). The position of azPhe on the protein surface was shown to have a significant effect on reaction yield and kinetics. Modification was used to install proteins with novel properties such as red-shifted fluorescence emission and the ability to bind to non-biological materials like graphene.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:585346 |
| Date | January 2013 |
| Creators | Reddington, Samuel C. |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/56226/ |
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