De novo Design of an Artificial bis-[4Fe4S] Binding Protein

Authors: Roy, A,, Sarrou, I., Vaughn, M.D., Astashkin, A.V., and Ghirlanda, G.
Title: De novo Design of an Artificial bis-[4Fe4S] Binding Protein
Source: Biochemistry
Year: 2013
Volume: 52
Pages: 7586–7594

ABSTRACT:

In nature, protein subunits containing multiple iron-sulfur clusters often mediate the delivery of reducing equivalents from metabolic pathways to the active site of redox proteins. The de novo design of redox active proteins should include the engineering of a conduit for delivery of electrons to and from the active site, in which multiple redox-active centers are arranged in a controlled manner. Here, we describe a designed three-helix protein, DSD-bis[4Fe4S], that coordinates two iron-sulfur clusters within its hydrophobic core. The design exploits the pseudo two-fold symmetry of the protein scaffold, DSD, which is a homodimeric three-helix bundle. Starting from the sequence of the parent peptide, eight leucine residues per dimer in the hydrophobic core are mutated to cysteine to provide the first coordination sphere for cubane-type iron-sulfur clusters. Incorporation of two clusters per dimer is readily achieved by in situ reconstitution, and imparts increased stability to thermal denaturation compared to the apo form of the peptide as assessed by CD monitored thermal denaturation. The presence of [4Fe4S] clusters in intact proteins is confirmed by UV-vis spectroscopy, gel filtration, analytical ultracentrifugation, and EPR spectroscopy. Pulsed electron-electron double resonance (ELDOR) experiments have detected the magnetic dipole interaction between the two clusters of about 0.7 MHz, which is consistent with the expected inter-cluster distance of 29-34 Å. Taken together, our data demonstrate the successful design of an artificial multi-FeS cluster protein with evidence of cluster-cluster interaction. The design principles implemented here can be extended to the design of multi-cluster molecular wires.


Date of online publication: Thu, 2013-10-03
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