title: De novo design of a non-natural fold for an iron-sulfur protein: alpha-helical coiled-coil with a four-iron four-sulfur cluster binding site in its central core.
abstract: Using a 'metal-first' approach, we computationally designed, prepared, and characterized a four-iron four-sulfur (Fe(4)S(4)) cluster protein with a non-natural alpha-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe(4)S(4) cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron-sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe(4)S(4) clusters in vitro. UV-Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, alpha-helical coiled-coil binding a single Fe(4)S(4) cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of low-potential, [Fe(4)S(4)](+) (S=1/2), with g values of g(zy)=(1.970, 1.975), and g(x)=2.053. The temperature, and power dependence of the signal intensity were also characteristic of [Fe(4)S(4)](+) clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as "wires" for coupling any given set of redox enzymes.
authors.affiliation: Plant Sciences Department, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Joanna
authors.initials: J
authors.lastname: Grzyb
authors.forename: Fei
authors.initials: F
authors.lastname: Xu
authors.forename: Lev
authors.initials: L
authors.lastname: Weiner
authors.forename: Eduard J
authors.initials: EJ
authors.lastname: Reijerse
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lubitz
authors.forename: Vikas
authors.initials: V
authors.lastname: Nanda
authors.forename: Dror
authors.initials: D
authors.lastname: Noy
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019295:Computational Biology; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017510:Protein Folding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism*; D014461:Ultracentrifugation
chemical_list: D007506:Iron-Sulfur Proteins; D013455:Sulfur; D007501:Iron