Genetically-encoded photoswitches for controlling apoptosis

Meah, Dilruba 2015. Genetically-encoded photoswitches for controlling apoptosis. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Light-Oxygen-Voltage (LOV) domains are flavoproteins that are part of photoreceptors found in plants, prokaryotes and algae. LOV domains act as biological switches in response sensors to oxygen, redox potential or light, making them ideal for use as an optogenetics tool. The protein-switch is activated by formation of a covalent adduct between the flavin cofactor and an internal cysteine residue, causing changes in the hydrogen bonding network in the core of the protein. This results in a conformational change in the LOV domain leading to undocking of an amphiphilic helix that is generally coupled to a catalytic or transcriptional activation domain. In this investigation the LOV2 domain of phototropin 1 from Avena sativa has been modified to control protein-protein interactions in apoptosis by incorporating the Bcl homology region 3 (BH3) of a key pro-apoptotic protein (Bid) to the mobile helix of AsLOV2 (J). The design, cloning, production and structure of these hybrid proteins (AsLOV-Bid1-4) are discussed and their photo-switching characteristics are examined using UV/Vis and CD spectroscopy. Half-lives of the proteins varied between 13 min and 7.5 min, with small deviation between UV/Vis and CD half-life measurements for each protein. A further investigation on the binding of AsLOV2-Bid proteins to Bcl-xL, the natural binding partner of Bid, has been conducted in the dark and light states using fluorescence anisotropy measurements. The results verified that AsLOV-Bid1-4 bound to Bcl-xL effectively in the light state with KD values at less than 300 nM. However, AsLOV-Bid2 also bound in the dark state with KD at 1 mM. A second LOV domain, YtvA from Bacillus subtilis is characterised using UV/Vis and CD spectroscopy. YtvA is a homodimeric photo-switch, with a longer relaxation half-life than Avena sativa LOV2, making it more suitable for use as an optogenetics tool. Here, investigations on amino acid residues key for the dimerisation of this protein were performed, before any hybrid proteins could be engineered. Residues V27 and I113, were replaced with aspartate residues by site-directed mutagenesis to explore the effects on the protein quaternary structure using size-exclusion chromatography. Comparing against standards mutant I113D is considered as monomeric, however further experiments need to be conducted to verify this.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Chemistry
Subjects:	Q Science > QD Chemistry
Date of First Compliant Deposit:	30 March 2016
Last Modified:	26 Jan 2024 16:23
URI:	https://orca.cardiff.ac.uk/id/eprint/70901

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