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Synthesis and DNA binding properties of peptides based on the bZip motif

Sheady, Sarah Elizabeth; (2000) Synthesis and DNA binding properties of peptides based on the bZip motif. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

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.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Synthesis and DNA binding properties of peptides based on the bZip motif
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; DNA-binding
URI: https://discovery.ucl.ac.uk/id/eprint/10102564
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