Novel routes to defined post translational modifications using non-canonical amino acids

Proteins are inherently limited by the properties of their constituent amino acids and attempt to overcome this by using post translational modifications (PTMs). PTMs are highly specific and can effectively modulate protein function faster than simple up or down regulation of protein production. However, PTMs often require a suite of other proteins to regulate and perform the modification to ensure accuracy, which can be hard to engineer into synthetic proteins. By introducing new chemistry into proteins via noncanonical amino acids (ncAAs) we can expand the range of new non-native PTMs that we can explore. Non-native PTMs (nnPTMs), have the potential to be both bioorthogonal and easily transferable between proteins. This thesis examines the effects of engineering nnPTMs into superfolder Green Fluorescent Protein (sfGFP) to study the effects on fluorescence of: 1) modification with small molecules (Chapter 3), 2) Creation of covalent protein dimers (Chapter4), 3) Interfacing proteins to carbon nanomaterials (Chapter 5), and 4) Look at the effects of engineering cooperativity using ncAAs (Chapter6). Most of this work focused on the ncAA, p-azido-L-phenylalanine (azF) as it has several properties that would be desirable for use in proteins such as photo reactivity and selective reactivity with alkynes. Moreover, as azF can be incorporated into any target protein in a range of hosts, it is an ideal starting point to engineer nnPTMs that are easily transferable. Throughout this thesis the importance of intricate hydrogen bonding networks and water channels, to the function of a protein, is made apparent through a range of in silico, structural and biophysical techniques. In silico modelling is used throughout to predict; the effects of nnPTMs on sfGFP structure (Chapter 3 and Chapter 6), dimer interfaces in Chapter 4, and show functional linking between sfGFP and carbon nanotubes in Chapter 5.