title: Subunit interaction in extracellular superoxide dismutase: effects of mutations in the N-terminal domain.
abstract: Human extracellular superoxide dismutase (hEC-SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC-SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X-ray structural determination have failed, the three-dimensional structure of hEC-SOD is unknown. This means that alternative strategies for structural studies are needed. The N-terminal domain of EC-SOD has already been studied using the fusion protein FusNN, comprised of the 49 N-terminal amino acids from hEC-SOD fused to human carbonic anhydrase (HCAII). The N-terminal domain in this fusion protein forms a well-defined three-dimensional structure, which probably contains alpha-helical elements and is responsible for the tetramerization of the protein. In this work, we have extended the studies, using site-directed mutagenesis in combination with size-exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic alpha-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction.
authors.affiliation: Department of Biochemistry, Umeå University, Sweden.
authors.forename: P
authors.initials: P
authors.lastname: Stenlund
authors.forename: D
authors.initials: D
authors.lastname: Andersson
authors.forename: L A
authors.initials: LA
authors.lastname: Tibell
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D013482:Superoxide Dismutase