Characterization of the heparin-binding domain of human extracellular superoxide dismutase.
The C-terminal, heparin-binding domain of human extracellular superoxide dismutase (hEC-SOD) has been studied as a fusion to human carbonic anhydrase II (HCAII). This technique allows the properties of the EC-SOD domain to be characterized. At the same time, it allows us to differentiate the contributions from the domain, from those properties originating from other parts of EC-SOD. The fusion of the 27 C-terminal amino acids of hEC-SOD to the C-terminal of HCAII (FusCC) resulted in the formation of a monomeric protein, which binds to heparin-Sepharose with approximately the same affinity as the tetrameric hEC-SOD. The structure of the fused C-terminal was characterized by CD and NMR spectroscopy and the data were compatible with the presence of alpha-helical structures as suggested by secondary structure predictions. The NMR data show that the C-terminal of FusCC moves independently from the rest of the protein and that its central part is involved in conformational exchange. The NOESY spectra demonstrate that the C-terminal in both FusCC and hEC-SOD binds to heparin, and that arginine side chains take part in the binding.
Tibell
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Department of Biochemistry, Umeå University, Sweden. lenat@chem.umu.se
Sethson
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Buevich
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space / Q000201:enzymology*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism*
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D006493:Heparin; D013482:Superoxide Dismutase; D002256:Carbonic Anhydrases