De Novo Design of a Non-Natural Fold for an Iron-Sulphur Cluster Proteins and Biophysical Characterization of Coiled-Coil Iron-Sulphur Protein-1 (CCIS-1)
2Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey, USA
Iron-sulphur [Fe-S] clusters proteins are metalloproteins that function as electron carriers, and play an important role in many biological redox processes. Those containing four-iron four-sulphur clusters are of interest to the development of alternative fuels such as molecular hydrogen. Our goal is to design artificial iron-sulphur cluster proteins that will serve as custom redox carriers by using computational tools. Developing an efficient system for assembling the [Fe-S] cofactors within the protein scaffold is critical to this endeavour. The computational design is achieved in multiple steps. The first step is to identify the [Fe-S] binding motif in the naturally occurring proteins. The selected motifs are then fitted with symmetry elements by constraining the conformation of the motif. The next step is to look for a stable backbone from the available libraries. The final step is to scan for optimal and stable co-factor assembly of the backbone and ligand. Coiled-Coil Iron-Sulphur protein-1 (CCIS-1) is one such example, designed as a minimal, non-natural protein scaffold to accommodate an active [Fe-S] cluster. Here, we report the heterologous expression of CCIS-1 in E. coli, and in-vivo assembly of [Fe-S] clusters within the CCIS holo-protein. This was achieved by supplementing iron and sulphur additives to the medium during the induction phase. To ensure the stable assembly of [Fe-S] clusters, the entire CCIS-1 purification is carried out strictly under anaerobic conditions inside a glove box. The assembled [Fe-S] clusters were characterised by chromatography, Mass Spectrometry (MS), optical spectroscopy, Circular Dichroism (CD), electrochemistry, Electron Paramagnetic Resonance (EPR) and Static Light Scattering techniques. Currently more proteins are being engineered, using various computational tools to accommodate a stable [Fe-S] cluster, within the protein scaffold.
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