The de novo design of a rubredoxin-like Fe site.
A redox center similar to that of rubredoxin was designed into the 56 amino acid immunoglobulin binding B1 domain of Streptococcals protein G. The redox center in rubredoxin contains an iron ion tetrahedrally coordinated by four cysteine residues, [Fe(S-Cys)4](-1),(-2). The design criteria for the target site included taking backbone movements into account, tetrahedral metal-binding, and maintaining the structure and stability of the wild-type protein. The optical absorption spectrum of the Co(II) complex of the metal-binding variant is characteristic of tetrahedral chelation by four cysteine residues. Circular dichroism and nuclear magnetic resonance measurements reveal that the metal-free and Cd(II)-bound forms of the variant are folded correctly and are stable. The Fe(III) complex of the metal-binding mutant reproduces the optical and the electron paramagnetic resonance spectra of oxidized rubredoxin. This demonstrates that the engineered protein chelates Fe(III) in a tetrahedral array, and the resulting center is similar to that of oxidized rubredoxin.
Farinas
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
Regan
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002104:Cadmium / Q000737:chemistry; D002942:Circular Dichroism; D003035:Cobalt / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry; D012416:Rubredoxins / Q000737:chemistry*; D013053:Spectrophotometry
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D008667:Metalloproteins; D011994:Recombinant Proteins; D012416:Rubredoxins; D002104:Cadmium; D003035:Cobalt; D007501:Iron; D003545:Cysteine