Determination of the structural role of the N-terminal domain of human extracellular superoxide dismutase by use of protein fusions.
The N-terminal domain, containing the 49 N-terminal amino-acid residues, of human extracellular superoxide dismutase (hEC-SOD) has been studied after construction of fusion proteins comprised of the defined domain and human carbonic anhydrase II (HCAII). The specific advantage of this technique is that it allows characterization of properties that are intrinsic to the N-terminal domain of hEC-SOD, i.e., the results are not obscured by properties pertaining to the rest of the hEC-SOD molecule. Moreover, the fusion to HCAII allows a rapid and gentle one-step purification by affinity chromatography. When the N-terminal domain was fused to the N-terminal of HCAII ( = FusNN) a well defined structure was formed and the resulting protein was tetrameric. When the same hEC-SOD-derived domain was fused to the C-terminal of HCAII ( = FusNC), no defined structure of the fused domain could be observed, and the resulting protein was monomeric. It was concluded that a 'free' N-terminus is required for formation of the proper structure of the N-terminal domain.
Department of Biochemistry, Umeå, Sweden.
L A
LA
Tibell
E
E
Skärfstad
B H
BH
Jonsson
D000595:Amino Acid Sequence; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013482:Superoxide Dismutase; D002256:Carbonic Anhydrases