Cardiff University | Prifysgol Caerdydd ORCA
Online Research @ Cardiff 
WelshClear Cookie - decide language by browser settings

Design and characterisation of an artificial DNA-binding cytochrome

Jones, D. Dafydd ORCID: and Barker, Paul D. 2004. Design and characterisation of an artificial DNA-binding cytochrome. ChemBioChem 5 (7) , pp. 964-971. 10.1002/cbic.200300569

Full text not available from this repository.


We aim to design novel proteins that link specific biochemical binding events, such as DNA recognition, with electron transfer functionality. We want these proteins to form the basis of new molecules that can be used for templated assembly of conducting cofactors or for thermodynamically linking DNA binding with cofactor chemistry for nanodevice applications. The first examples of our new proteins recruit the DNA‐binding basic helix region of the leucine zipper protein GCN4. This basic helix region was attached to the N and C termini of cytochrome b562 (cyt b562) to produce new, monomeric, multifunctional polypeptides. We have fully characterised the DNA and haem‐binding properties of these proteins, which is a prerequisite for future application of the new molecules. Attachment of a single basic helix of GCN4 to either the N or C terminus of the cytochrome does not result in specific DNA binding but the presence of DNA‐binding domains at both termini converts the cytochrome into a specific DNA‐binding protein. Upon binding haem, this chimeric protein attains the spectral characteristics of wild‐type cyt b562. The three forms of the protein, apo, oxidised holo and reduced holo, all bind the designed (ATGAcgATGA) target DNA sequence with a dissociation constant, KD, of approximately 90 nM. The protein has a lower affinity (KD ca. 370 nM) for the wild‐type GCN4 recognition sequence (ATGAcTCAT). The presence of only half the consensus DNA sequence (ATGAcgGGCC) shifts the KD value to more than 2500 nM and the chimera does not bind specifically to DNA sequences with no target recognition sites. Ultracentrifugation revealed that the holoprotein–DNA complex is formed with a 1:1 stoichiometry, which indicates that a higher‐order protein aggregate is not responsible for DNA binding. Mutagenesis of a loop linking helices 2 and 3 of the cytochrome results in a chimera with a haem‐dependent DNA binding affinity. This is the first demonstration that binding of a haem group to a designed monomeric protein can allosterically modulate the DNA binding affinity.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Biosciences
Publisher: Wiley
ISSN: 1439-4227
Last Modified: 09 Nov 2022 10:23

Citation Data

Cited 13 times in Scopus. View in Scopus. Powered By Scopus® Data

Actions (repository staff only)

Edit Item Edit Item