Design and Characterisation of an Artificial DNA‐Binding Cytochrome

Abstract

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 b(562) (cyt b(562)) 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 b(562). The three forms of the protein, apo, oxidised holo and reduced holo, all bind the designed (ATGAcgATGA) target DNA sequence with a dissociation constant, K(D), of approximately 90 nM. The protein has a lower affinity (K(D) ca. 370 nM) for the wild-type GCN4 recognition sequence (ATGAcTCAT). The presence of only half the consensus DNA sequence (ATGAcgGGCC) shifts the K(D) 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.

Controlling Self‐Assembly by Linking Protein Folding, DNA Binding, and the Redox Chemistry of Heme

“…It has been observed that the presence of only one BHR region attached to either the N or C terminus of the intact cyt b 562 does not promote tight, specific DNA binding. [20] However, the increase in helical content of the fragments in the presence of DNA (Supporting Information) suggests that both the bhrN12 and 34Cbhr fragments bind to DNA alone. This crucial difference should allow DNA to direct the assembly and so influence heme binding.…”

“…Titration of bhrN12 and 34Cbhr with ferric heme (2 mm) revealed a low affinity for the cofactor (Figure 2 a); K D > 27000 nm AE 3000 is at least 1000-fold higher than that observed for the intact DBC. [20] Under reducing conditions, the bhrN12 and 34Cbhr fragments bind heme with an affinity (K D = 435 nm AE 85 nm) much lower than that of the oxidized form (Figure 2 b). In both oxidation states, the spectrum was identical to that of wild-type cyt b 562 , and the DBC was observed on the addition of 5 equivalents of bhrN12 and 34Cbhr (relative to heme), indicating that the helical bundle had assembled correctly (Supporting Information).…”

“…The original design of the single-molecule scaffold [20] has the BHR of GCN4, attached to the termini of the four-helix bundle of cyt b 562 , thus creating a DBC. The design specifically arranges the conducting material (heme) on a template (DNA), and events that occur at the heme center can be transmitted to the DNA-binding region and vice versa, which Figure 1.…”

“…[20] This design created a protein capable of being assembled on a designated template (DNA) that could potentially both carry and control current flow. The novel DNA-binding cytochrome (DBC) exhibits spectral characteristics and heme affinity that is comparable to those of the parent cyt b 562 and also has the ability to bind DNA sequences based around those recognized by GCN4.…”

“…To determine if DNA can influence complex assembly, the optimum double-stranded DNA sequence for the chimera [20] dsNC1 was employed to maximize potential binding. It has been observed that the presence of only one BHR region attached to either the N or C terminus of the intact cyt b 562 does not promote tight, specific DNA binding.…”