Abstract
Cu-metallothionein was purified from Saccharomyces cerevisiae harboring plasmids containing mutated CUP1 metallothionein genes resulting in deletions at the carboxy-terminal end of the polypeptide. The truncated polypeptides are recovered as polypeptides of 35 and 48 residues in length. The Cu-S cluster in the wild-type metallothionein and the two truncates were characterized. The truncated proteins, designated T35 and T48, contain 4 and 2 fewer cysteinyl residues, respectively, compared to the 12 cysteines in wild-type metallothionein; yet the mutant molecules bind Cu(I) ions in a stoichiometry comparable to the wild-type protein, i.e. 7-8 mol eq. The Cu(I) ions bound to T48 are as tenaciously bound as those bound to the wild-type molecule. The electronic transitions in the ultraviolet are similar for Cu-T48 and the wild-type protein. Both mutants and wild-type Cu-protein exhibit luminescence. The corrected emission maxima occurs at 609 nm with a corrected excitation peak near 277 nm. The luminescence quantum yield and lifetime of fluorescence decay of Cu-T48 and wild-type Cu-metallothionein are similar. The absolute quantum yield of the wild-type Cu-protein luminescence is 0.0058 and has a 440-ns lifetime. The similar fluorescence rate constant in the two molecules suggests they possess a similar chromophore. The Cu-T35 protein is more labile than Cu-T48 or the wild-type protein in the association of Cu(I) ions and the air sensitivity of the electronic transitions and luminescence. Although T48 lacks 2 of the 12 cysteines in the wild-type protein, we are unable to detect any differences in the properties of the native metal clusters in the two molecules; T35 lacking 4 cysteinyl residues forms a Cu(I) cluster with properties significantly different from the wild-type molecule. Properties of the Cu-thiolate cluster were also studied in Cu(I)-reconstituted samples. The cluster in wild-type metallothionein forms in all-or-nothing fashion. This conclusion is based on copper binding stoichiometry and luminescence studies. The relative quantum yield of samples with intermediate Cu(I) levels was constant, consistent with all-or-none cluster formation.
Highlights
Cu-metallothionein was purified fromSaccharomy- Metallothioneinhas beenpurifiedfromdiversesources ces cerevisiae harboring plasmids containing mutated since its first isolation from equine renal cortex (1-3)
Le. 7-8 mol eq The Cu(1) ions bound to T48 are as function in some aspect of zinc and copper metabolism in tenaciously bound asthose bound to thewild-type mol- additiontotheirclearcapacityinmetal detoxification
Within each domain the structure consists of the metal cluster core wrapped by a polypeptide monolayer ( 5 ) .The metal ions arethermodynamically stable but exhibitfacile exchange with freemetal ions orwith metal ions bound to other metallothioneinmolecules (8)
Summary
Vol 263, No., Issue of May 15, pp. 6688-6694,1988 Printed in U.S.A. Characterization of the Copper-Thiolate Clusterin Yeast Metallothionein and Two Truncated Mutants*. The electronic transitions in the ultravioleat re similar for Cu-T48 and the wild-type protein. The luminescence quantum yield and lifetime of fluorescence decay of Cu-T48 and wild-type Cu-metallothionein are similar. Than Cu-T48 or the wild-type protein in thaessociation It is clear that mammalian metallothionein bindsCu(1) in a of Cu(1) ions and the air sensitivity of the electronic configuration and stoichiometry differenftrom Zn(I1). The relative quantum iue is classed as a metallothionein in having the mentioned yield of samples with intermediate Cu(1) levels was properties and exhibiting limihtoedmology to the mammalian constant, consistent with all-or-none cluster forma- protein (10, 11).The yeast metallothionein gene encodes a tion. Are missing either 2 or 4 of themetal ligatingcysteinyl residues In this reporwte present results on the characterization of the Cu-thiolate cluster in yeast metallothionein and two truncated mutants.
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