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Title: Synthesis and DNA binding properties of peptides based on the bZip motif
Author: Sheady, Sarah Elizabeth
ISNI:       0000 0001 3401 7167
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2000
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GCN4 is a transcriptional activator, found in S. Cerevisiae, that belongs to the well characterised bZip family of DNA binding proteins. The protein is 281 residues in length with the bZip portion comprising the C-terminal 60 residues. The bZip portion of the protein can be divided into two distinct regions, the leucine zipper region that is responsible for dimerisation through formation of a parallel coiled coil structure and the basic region that is responsible for contacting DNA in a sequence specific fashion. Work from many research groups has shown that it is possible to chemically dimerise the basic region and maintain specificity. In an effort to extend this work peptide 1 was designed and synthesised (Figure 1). Figure 1 - Peptide 1 [diagram] Peptide 1 contains the minimum number of residues required to specifically bind to the native binding site with an intercalator, attached via a linker to the N-terminal, to increase affinity. Attempted determination of the specificity of binding using gel retardation based on in vitro selection proved inconclusive, with difficulties arising in both the selection and amplification events. Using gel retardation assays, peptide 1 was found to bind specifically to the AP1 with a Kd of 25±6pM, and to the CREB/ATF sites, with a Kd of 28±8pM. This indicates that the original design of the peptide to produce a peptide with retained specificity and increased affinity has been fulfilled. The addition of the intercalator also produced non specific binding of the peptide monomer (pepfide 8, Figure 2). Figure 2 - Peptide 8 [diagram] In an effort to further enhance binding affinity, a series of peptides were designed based on peptide 1 that contained different intercalator/linker systems. The required intercalator/linker systems have been synthesised and attempts made to couple these to peptides.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available