DNA polymerases from hyperthermophiles.
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K B Nielson
Publications and source records attributed to K B Nielson.
Yeast metallothionein. Sequence and metal-binding properties.
The protein product of the CUP1 locus in Cu-resistant Saccharomyces cerevisiae has been purified and characterized. The protein was found to lack the first 8 amino acids predicted by the nucleotide sequence of the gene. The residues removed from the amino-terminal region include 5 hydrophobic residues, two of which are aromatic. The unique amino terminus starting at Gln9 of the putative DNA translation product was observed for metallothionein purified in the presence of various protease inhibitors or from a pep4 mutant yeast strain deficient in vacuolar proteases. The remainder of the primary structure of the protein is equivalent to the decoded DNA sequence, so yeast metallothionein is a 53-residue polypeptide of molecular weight 5655. The isolated protein contained 8 copper ions ligated by 12 cysteines/molecule. Reconstitution studies of the apo-molecule revealed that 8 mol eq of Cu(I) conferred maximal stability against proteolysis and depleted the zinc content of zinc-saturated metallothionein. These assays suggested that the protein has 8 binding sites for Cu(I). Ag(I) ions bound to the protein with the same stoichiometry. Yeast metallothionein was also observed to coordinate Cd(II) and Zn(II) ions in vitro. In studies of direct binding, protection against proteolysis, and metal ion exchange, these divalent ions were found to associate with the protein with a maximal stoichiometry of 4 ions/molecule. Yeast metallothionein thus exhibits two distinct binding configurations for Cu(I) and Cd(II) as does the mammalian protein.
Amino Acid Sequence↗
Independence of the domains of metallothionein in metal binding.
Mammalian metallothionein is a low molecular weight protein with two metal-binding domains. To determine if metal binding in one domain affects binding in the other, we prepared peptides corresponding to the regions that enfold the two metal-thiolate clusters. Metal reconstitution studies of these peptides revealed stoichiometries of metal binding similar to those observed within the intact molecule. Thus, the alpha domain coordinates 4 Cd(II), 6 Cu(I), or 6 Ag(I) ions regardless of whether the domain is part of the total protein or is studied as a separate peptide. Likewise, the beta domain binds 3 Cd(II), 6 Cu(I), or 6 Ag(I) ions in both the intact protein and as a separate peptide. If cluster B in intact metallothionein is preformed with Cu(I) or Ag(I), cluster A saturates with either 4 mol eq of Cd(II) or 6 mol eq of Ag(I). Similarly, preformation of the A cluster with Cd(II) does not affect the binding of 6 Cu(I) ions in the B cluster. Therefore, the metal-dependent folding of the protein to create one cluster occurs independent of constraints or influences from the other domain. Formation of the protein with a tetrahedrally coordinated metal in one cluster and a trigonally coordinated metal in the other center is possible.
Cadmium↗
Distinct metal-binding configurations in metallothionein.
In a study of the binding stoichiometry of various metals to rat liver metallothionein, the protein appears to coordinate metals in 2 distinct configurations. Ions of at least 18 different metals were shown to associate with the protein suggesting that there is little specificity in binding. Most metals exhibited saturation binding at 7 mol eq forming M7-metallothionein. These included Bi(III), Cd(II), Co(II), Hg(II), In(III), Ni(II), Pb(II), Sb(III), and Zn(II). Others metals including Os(III), Pd(II), Pt(IV), Re(V), Rh(III), and Tl(III) give a positive indication of binding, but stoichiometries were unclear. Ag(I) and Cu(I) bound in clusters as M12-metallothionein. This binding stoichiometry was determined in 3 ways: (a) by determining the equivalence point in Cu- and Ag-titrated samples where resistance to proteolysis is maximal; (b) by determining the point where Zn ions are completely displaced from Zn7-metallothionein; and (c) by direct binding studies. Ag-reconstituted protein, recovered from gel filtration, had an average Ag content of 11.5 g atoms/mol of protein. A similar stoichiometry for the Cu-protein resulted from displacement of Zn from Zn7-metallothionein by Cu(I). The M12-protein was converted to the M7-protein by displacement of Ag(I) or Cu(I) with 7 mol eq of Hg(II). Whereas the distribution of metals in the 2 domains of M7-metallothionein is M4 alpha and M3 beta, the arrangement in the M12-molecule is probably M6 alpha and M6 beta. We propose that metallothionein ligates Ag(I) and Cu(I) in a trigonal geometry by bridging thiolates. This is in contradistinction to a tetrahedral binding geometry in the M7-protein. Distinct binding configurations may result in different tertiary structures for M7- and M12-proteins which may relate to metabolic specificity of Zn-metallothionein and Cu-metallothionein, respectively.
Animals↗
Structural characterization of the isoforms of neonatal and adult rat liver metallothionein.
Metallothionein was purified from the livers of adult and neonatal rats. The complete amino acid sequences of isoforms I and II of Cd-induced adult metallothionein were determined by automated Edman degradation of CNBr and tryptic peptides of carboxymethylated proteins. Both isoproteins contain 61 residues, but differ at 12 of those positions. The positions of the 20 cysteinyl residues are invariant with respect to those in other known mammalian metallothioneins. Based on the following criteria, the two soluble and constitutive Zn-metallothionein isoforms from neonatal liver appear identical to their corresponding forms in the adult animal: 1) they behave identically on reverse-phase high-pressure liquid chromatography, anion-exchange chromatography and nondenaturing gel electrophoresis; 2) their amino acid compositions are the same within experimental error; and 3) their amino acid sequences are identical in the first 23 residues, even though isoforms I and II differ in 7 of those positions. It appears highly likely that isoforms from both neonatal and adult rats are encoded by the same genes, and therefore that the large age-dependent concentration differences seen in the liver must be due to variation in gene regulation.
Age Factors↗
Preferential binding of copper to the beta domain of metallothionein.
Proteolytic studies of rat liver metallothionein reconstituted in vitro with Cu salts revealed that the 2 metal centers fill in an ordered fashion. The B cluster in the NH2-terminal beta domain fills prior to Cu binding in cluster A. This is in contradistinction to cluster formation induced by the binding of Cd or Zn ions in which cluster A is the center of initial binding. The formation of metal cluster B by Cu occurs in a cooperative fashion yielding a saturated cluster with approximately 6 Cu+ ions bound. The B cluster is saturated with Cd or Zn after binding of only 3 metal ions. The preferential binding of Cd and Cu to the alpha and beta domains, respectively, and the tolerance toward proteolysis of these 2 different half saturated molecules permit the isolation of each domain. The metal cluster in each isolated domain can be reversibly formed with predicted stoichiometries of Cd and Cu. The folding of the polypeptide therefore appears to create each cluster independently. The metal binding data suggest that Cu-metallothionein contains 11-12 Cu ions, 6 bound in the beta domain and 5-6 in the alpha domain. In contrast, Cd-metallothionein contains 7 Cd ions, 3 bound to beta and 4 to alpha.
Amino Acids↗
Formation of the metal-thiolate clusters of rat liver metallothionein.
The isoforms of rat liver apo-metallothionein (MT) were reconstituted in vitro with Cd and Zn ions to study the order of binding of the seven metal sites. Reconstitution with seven Cd ions resulted in a metalloprotein similar to induced Cd,Zn-MT by the criteria of electrophoretic mobility, insensitivity to proteolysis by subtilisin and the pH-dependent release of Cd. Proteolytic digestion of MT reconstituted with sub-optimal quantities of Cd followed by separation of Cd-containing polypeptide fragments by electrophoresis and chromatography revealed metal ion binding initially occurs in cluster A. Upon saturation of the four sites in cluster A, binding occurs in the three metal center, cluster B. Samples reconstituted with one to four Cd or Zn ions per protein molecule, followed by digestion with subtilisin, yielded increasing amounts of a proteolytically stable polypeptide fragment identical with the alpha fragment domain encompassing the four metal center. Samples renatured with five to seven Cd ions per MT molecule showed decreasing quantities of alpha fragment and increasing amounts of nativelike MT. The binding process in each domain is cooperative. Reconstitution of apo-MT with two Cd ions followed by proteolysis yields a 50% recovery of saturated Cd4-alpha cluster. Likewise, when Cd5-renatured MT was digested with subtilisin, 30% of the molecules were identified as Cd7-MT with the remainder as Cd4-alpha fragment.
Animals↗
Order of metal binding in metallothionein.
Purified isoforms of rat liver apometallothionein were reconstituted in vitro with Cd and Zn ions to study the order of binding of the 7 metal sites in the two separate metal clusters, one containing four metal ions (cluster A) and the other containing three (cluster B). Reconstitution with 7 Cd ions resulted in a metalloprotein similar to induced Cd,Zn-metallothionein by the criteria of electrophoretic mobility, insensitivity to proteolysis by subtilisin, and the pH-dependent release of Cd. Proteolytic digestion of metallothionein reconstituted with suboptimal quantities of Cd followed by separation of Cd-containing polypeptide fragments by electrophoresis and chromatography revealed metal ion binding initially occurs in the 4-metal center, cluster A. Upon saturation of the 4 sites in cluster A, binding occurs in the 3-metal center, cluster B. Samples reconstituted with 1 to 4 Cd ions per protein molecule, followed by digestion with subtilisin, yielded increasing amounts of a proteolytically stable polypeptide fragment identical with the alpha fragment domain that is known to encompass the 4-metal center. Samples renatured with 5 to 7 Cd ions per metallothionein molecule showed decreasing quantities of alpha fragment and increasing amounts of native-like metallothionein. Similar results were obtained in reconstitution studies with Zn ions. Samples reconstituted with 7 Cd eq followed by incubation with EDTA revealed that cluster B Cd ions were removed initially. The binding process in each domain is cooperative. Reconstitution of apometallothionein with 2 Cd ions followed by proteolysis yields a 50% recovery of saturated Cd4 alpha cluster. Likewise, when Cd5-renatured metallothionein was digested with subtilisin, 30% of the molecules were identified as Cd7 metallothionein with the remainder as Cd4 alpha fragment.
Amino Acids↗