Using a reprogrammed genetic code to modulate protein activity by novel post-translational control

Hartley, Andrew M 2014. Using a reprogrammed genetic code to modulate protein activity by novel post-translational control. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Despite the diverse structures and functions sampled by the proteome, all proteins comprise 20 canonical amino acids that sample only a small percentage of available chemistry. This limitation is lifted somewhat through the use of post-translational modifications, however the limit imposed by the restricted number of amino acids inherently limits the variety of protein function and control that can be accessed. One powerful route to diversify the chemistry sampled by proteins is through genetically encoded unnatural amino acid (uAA) incorporation. The uAA p-azido-L-phenylalanine (AzPhe) can introduce two novel methods of control, photochemical covalent rearrangement and Click chemistry. AzPhe incorporation combined with these two methods of novel post-translational control were used to modulate the function of two distinct proteins; TEM β-lactamase and sfGFP. This thesis introduces the use of uAAs and the technical modifications required to enable uAA incorporation in vivo. It describes the in silico approach taken to evaluate potential mutations based on the likelihood of them imparting novel changes to protein function. Nine positions in TEM β-lactamase were chosen for uAA incorporation and the effect on activity was then determined using kinetic analyses. AzPhe incorporation alone resulted in a variety of effects on enzyme activity, ranging from small increases to complete loss of activity. Subsequent post-translational modification using UV light resulted in only slight changes in activity. Modification via Click chemistry using dibenzyl cyclo-octyne (DBCO) derivatives resulted in either inhibition or increased catalytic activity, depending on the position of AzPhe incorporation and the type of adduct used. Click chemistry was then used to modify TEM β-lactamase with other chemical modifications that enable the immobilization of proteins onto two different surfaces. The π-π stacking interaction between a DBCO-pyrene moiety and graphene was exploited to attach TEM β-lactamase to graphene in a defined and controlled manner, placing the active site in close proximity to the electron cloud of the sp2-bonded material. TEM β-lactamase was then modified using two DNA oligonucleotides that define assembly of a DNA origami “tile”. DNA origami can be used to immobilize multiple proteins at several defined positions, enabling the re-creation of enzyme pathways or signalling cascades in vitro. Finally, AzPhe was incorporated into sfGFP and the effects of its incorporation and subsequent modification on fluorescence were explored. The incorporation of AzPhe resulted in a blue shifted λmax, a change that was reversed upon UV irradiation. X-ray crystallography suggested that a hydrogen-bonding network involving the chromophore and surrounding residues was disrupted upon AzPhe incorporation, but then reformed upon modification of the uAA. Click chemistry had a variable effect on fluorescence depending on the modification used. Modification of AzPhe with a large fluorescent dye had no effect on the sfGFP fluorescence spectrum, but enabled FRET between the two chromophores. Modification with a DBCO-amine had the same effect as UV irradiation. Overall, this thesis has shown that the use of genetically encoded uAA incorporation coupled with novel post-translational modifications is a powerful approach for modifying protein function, and facilitating defined interfacing with new and useful materials.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > QH Natural history > QH426 Genetics
Funders:	BBSRC
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Oct 2023 10:51
URI:	https://orca.cardiff.ac.uk/id/eprint/68901

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