Towards visible light switching of peptide-DNA and peptide-protein interactions

Dean, Ryan 2016. Towards visible light switching of peptide-DNA and peptide-protein interactions. PhD Thesis, Cardiff University. Item availability restricted.

Preview	PDF - Accepted Post-Print Version Available under License Creative Commons Attribution Non-commercial No Derivatives. Download (8MB) | Preview
	PDF - Supplemental Material Restricted to Repository staff only Download (667kB)

Abstract

Peptides derived from DNA-binding zinc finger proteins were synthesised with pairs of cysteine residues with i,i+7 and i,i+11 relative spacings introduced into their sequence. The sidechains of these cysteine residues were then alkylated with the well-known water soluble photochrome 3,3’-bis(sulfo)-4,4’-bis(chloroacetamino) azobenzene (BSBCA). The change of shape of the azobenzene dye in these peptide-dye conjugates allowed photocontrol of peptide structure and thus peptide-DNA interactions. For a single zinc finger helix, UV irradiation yielded a peptide conjugate with a dissociation constant with respect to its cognate DNA sequence of 100 nM with no binding apparent prior to irradiation. However, the relatively short half-life of BSBCA proved a stumbling block, particularly in the control of larger peptides using multiple azobenzenes to control several -helical structural elements within large peptides. In addition to the short half-life of cis-BSBCA under physiological conditions, multiple BSBCA switches attached to the same peptide were shown not to relax independently of each other. These results led to the design, synthesis and examination of novel photo switches sensitive to visible, rather than UV light, with improved light state half-lives and bidirectional optical switching. Initial studies on thioindigo-based switches proved that molecules sufficiently polar to be water soluble were inaccessible by concise synthetic routes. Attention was then turned to the synthesis of ortho-halogen substituted azobenzenes and investigation of several new conjugation strategies for linking these photosensitive molecules to peptides. Subsequent refinements to the design of the tetra-ortho-halogen substituted azobenzenes altered the position of UV/visible absorbance bands of the cis and trans isomers to create a 47 nm separation in the wavelengths of the n-π* absorbances of the isomers to allow effective bidirectional switching. These changes also improved the half-life of the cis state from 24 minutes at 20 0C to 3,256 minutes at 60 0C. One of these new azobenzenes was reacted with apoptosis-inducing Bak peptides with different cysteine spacings (i,i+7 and i,i+11). Less stringent control over the binding of these peptides to Bcl-xL was observed than with BSBCA, perhaps due to the more flexible nature of the new crosslinker, but the optical properties of this class of molecules suggest a little further development will yield much improved photoswitches.

Item Type:	Thesis (PhD)
Date Type:	Submission
Status:	Unpublished
Schools:	Chemistry
Subjects:	Q Science > QD Chemistry
Date of First Compliant Deposit:	29 May 2018
Last Modified:	11 Dec 2020 02:36
URI:	https://orca.cardiff.ac.uk/id/eprint/111805

Actions (repository staff only)

Edit Item