title: Separation and characterization of the metal-thiolate-cluster domains of recombinant sea urchin metallothionein.
abstract: Partial metal depletion and 113Cd-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylocentrotus purpuratus (Cd7-MTA) binds its metal ions in a four-metal (Cd4Cys11) and a three-metal (Cd3Cys9) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wang, Y., Mackay, E.A., Zerbe, O., Hess, D., Hunziker, P.E., Vasak, M. & Kägi, J.H.R. (1995) Biochemistry 34,7460-7467]. This partitioning has now been confirmed by bisecting native Cd7-MTA with subtilisin into products bearing only a single metal-thiolate cluster. Their separation by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N-terminal polypeptides containing 37-39 residues with four Cd ions and a set of C-terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster-harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal-bound cysteine residues of the C-terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three-metal cluster was lost to the N-terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N-terminal domain retained the CD profile of the four-metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three-metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd7-MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the three-metal cluster.
authors.affiliation: Biochemisches Institut, Universität Zürich, Switzerland.
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: D
authors.initials: D
authors.lastname: Hess
authors.forename: P E
authors.initials: PE
authors.lastname: Hunziker
authors.forename: J H
authors.initials: JH
authors.lastname: Kägi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000737:chemistry*; D002614:Chelating Agents / Q000494:pharmacology; D002942:Circular Dichroism; D004492:Edetic Acid / Q000494:pharmacology; D013058:Mass Spectrometry; D008668:Metallothionein / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D012617:Sea Urchins / Q000737:chemistry*; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D002614:Chelating Agents; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D002104:Cadmium; D008668:Metallothionein; D004492:Edetic Acid