Information on Metal Binding Properties of Metallothioneins from Optical Spectroscopy

Stillman, Martin J.; Law, Annie Y.C.; Cai, Wuhua; Żelazowski, Andrzej J.

doi:10.1007/978-3-0348-6784-9_13

Cited by 48 publications

(55 citation statements)

References 8 publications

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“…Electronic transitions that occur within this field will be optically active (and will therefore exhibit a CD spectrum). Due to the fortuitous absence of aromatic amino acids in this protein, the ultravioletvisible CD spectrum of metallothioneins above 230 nm is entirely metal dependent, usually involving 'Cys'-S-+ metal charge-transfer transitions [31,33,34,401. Replacing one metal with another will have two effects on the CD signal.…”

Section: Resultsmentioning

confidence: 99%

“…Metal concentrations were determined with a Varian 875 atomic absorption spectrometer. The ratios of the concentrations of Zn, Cu, and in some cases Cd, were used to determine the metal-protein stoichiometries, based on an initial binding of 7 Zn(II)/metallothionein molecule [34]. The spectral data were organized and plotted on an HP 7550A plotter with the spectral database programs Spectra Manager [39], and Plot3D (Gasyna, Z. and Stillman, M. J., unpublished program).…”

Section: Methodsmentioning

confidence: 99%

“…5 ) occurs in two steps [34]. The first four Cd2+ results in a red shift of the CD band maximum from 240 nm in Zn,-metallothionein to 251 nm in Cd,Zn,-metallothionein ( Fig.…”

Section: Formation Of Mixed-metal (Cuzn-and Cucd-) Metallothioneinsmentioning

confidence: 95%

“…At low temperatures the kinetic product may be long lived and its presence may dominate subsequent reactions. For metallothionein, different cluster structures may be exhibited by the kinetic and thermodynamic product [13,34,351. Fig.…”

Section: Cu' Binding To Zn-metallothioneinmentioning

confidence: 99%

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Copper Binding to Rabbit Liver Metallothionein

Presta

Green

Żelazowski

et al. 1995

European Journal of Biochemistry

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Circular dichroism and ultraviolet absorption spectral data have been used to probe the binding mechanism for formation and the structure of the copper(1)-thiolate binding clusters in rabbit liver metallothiionein during addition of Cu' to aqueous solutions of Zn,-metallothionein 2 and Cd,Zn,-metallothionein 2. Mammalian metallothionein binds metals in two binding sites, namely the a and P domains. Spectral data which probe the distribution of Cu(1) between the two binding domains clearly show that both the site of binding (a or p), and the structures of the specific metal-thiolate clusters formed, are dependent on temperature and on the nature of the starting protein (either Zn,-metallothionein or Cd,Zn,-metallothionein). CD spectra acquired during the addition of Cu' to Zn,-metallothionein show that Cu' replace the bound Zn(I1) in a domain-distributed manner with complete removal of the Zn(I1) after addition of 12 Cu'. Spectral and metal analyses prove that a series of Cu(1)-metallothionein species are formed by a non-cooperative metal-binding mechanism with a continuum of Cu(1) :metallothionein stoichionietries. Observation of a series of spectral saturation points signal the formation of distinct optically active Cu(1)-thiolate structures for the Cu,Zn,-metallothionein, Cu,,-metallothionein, and the Cu,,-metallothiionein species. These data very clearly show that for Cu(1) binding to Zn,-metallothionein, there are sleveral key Cu(1):metallothionein stoichiometric ratios, and not just the single value of 12. The CD spectra up to the Cu,,-metallothionein species are defined by bands located at 255(+) nm and 280(--) nm. Interpretation of the changes in the CD and ultraviolet absorption spectral data recorded between 3°C and 52°C as Cu' is added to Zn-metallothionein show that copper-thiolate cluster formation is strongly temperature dependent. These changes in spectral properties are interpreted in terms of kinetic versus thermodynamic control of the metal-binding pathways as Cu' binds to the protein. At low temperatures (3 "C and 10OC) the spectral data indicate a kinetically controlled mechanism whereby an activation barrier inhibits formation of ordered copper-thiolate structures until formation of Cu,,-metallothionein. At higher temperatures (>3OoC) the activation barrier is overcome, allowing formation of new Cu(1)-thiolate clusters with unique spectral properties, especially at the Cu,Zn,-metallothionein point. The CD spectra also show that a Cu,,-metallothionein species with a well-defined, three-dimensional structure forms at all temperatures, characterized by a band near 335 nm, indicating the presence of digonal Cu(1). Complicated CD spectral changes are observed when Cu' is added to Cd,Zn,-metallothionein. The spectral data are interpreted in terms of domain-distributed binding followed by rearrangement to form the domain-specific product. In the Cu,Cd,-metallothionein species, the Cu' are ultimately bound specifically to the p domain of the protein. This complex is characterised by the CD spectrum o...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…5 ) occurs in two steps [34]. The first four Cd2+ results in a red shift of the CD band maximum from 240 nm in Zn,-metallothionein to 251 nm in Cd,Zn,-metallothionein ( Fig.…”

Section: Formation Of Mixed-metal (Cuzn-and Cucd-) Metallothioneinsmentioning

confidence: 95%

Section: Cu' Binding To Zn-metallothioneinmentioning

confidence: 99%

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Copper Binding to Rabbit Liver Metallothionein

Presta

Green

Żelazowski

et al. 1995

European Journal of Biochemistry

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“…Apoprotein was prepared by exposure to anaerobic 0.1 M HCl as described previously (12) and separated from free cadmium by G-25 size-exclusion Sephadex chromatography. The concentration of apoprotein was determined by amino acid analysis (7), and the reduction state of cysteine residues was assessed in a 2,2Ј-dithiobis(5-nitropyridine) assay (13). Proteins were resolved by SDS-polyacrylamide gel electrophoresis (14).…”

Section: Methodsmentioning

confidence: 99%

Metal Ion Trafficking in Earthworms

Stürzenbaum

Winters

Galay

et al. 2001

Journal of Biological Chemistry

143

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Exposure to cadmium poses a considerable risk to human health and environmental safety. Earthworms reside in the most contaminated sites on earth, displaying a phenomenal tolerance to toxic heavy metals. They exhibit a distinct metabolic pathway that allows the bio-accumulation of cadmium to yield body burdens in excess of 1/1000th of total dry body weight, a most impressive figure by any standard. However, the precise molecular mechanism underlying this phenomenon remains to be unraveled. This study meets this challenge by fully characterizing the major metal-binding protein in earthworms, namely the two isoforms of metallothionein. Chemical analysis of recombinant protein showed that although both isoforms bind equimolar amounts of cadmium (6 mol), wMT-2 is more stable during proton competition. Furthermore, isoform-specific transcript analysis demonstrated that only wMT-2 is responsive to cadmium in a dose and temporal manner. The specific sequestration of cadmium to wMT-2 protein was confirmed in situ using polyclonal antisera. The latter also provided the means for mapping the cellular and intracellular distribution of metallothionein, thus yielding a holistic insight into its involvement in cadmium transit during absorption, storage, and excretion. The structure-function relationship of wMT-2 and its role in cadmium detoxification through sequestration and compartmentalization is discussed.Earthworms are central to soil quality and fertility. The first authoritative account of the central role of the earthworm in soil formation, quality, and fertility dates back to Charles Darwin well over 100 years ago (1). More recent studies have shown this oligochaete annelid exhibiting an exceptional tolerance to cadmium, residing in soils with concentrations exceeding 600 g of cadmium/g dry weight (2). In addition to surviving this toxicological challenge, they bio-accumulate this metal ion to a body burden in excess of 1 mg/g dry weight (2, 3). This accumulation of cadmium is in stark contrast to the exclusion observed with other metal ions, for example, copper (4). However, even where ions with closely related chemistry, such as cadmium and zinc, are elevated within the same environment, the compartmentalization, and therefore the metabolic pathway, remains distinct (5). Therefore, the earthworm must have a highly developed and specific trafficking pathway for this toxic metal ion. Ultrastructural mapping has shown that the majority of the cadmium is sequestered as a thiol-based bioinorganic complex within intracellular vesicles in the earthworm's primitive liver-like tissue, the chloragog (5).The co-localization of cadmium and sulfur within a distinct intracellular organelle suggested the involvement of a sulfurrich protein. Metallothioneins (MTs) 1 are cysteine-rich cationic proteins that have been linked to cadmium detoxification in a wide range of phylogenetic orders (e.g. humans (6), fish (7), Caenorhabditis elegans (8), springtails (9), and others). However, until recently (2) no molecular information has b...

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Metallobiological Necklaces: Mass Spectrometric and Molecular Modeling Study of Metallation in Concatenated Domains of Metallothionein

Chan¹,

Huang²,

Watt

et al. 2008

Chemistry A European J

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The ubiquitous protein metallothionein (MT) has proven to be a major player not only in the homeostasis of Cu(I) and Zn(II), but also binds all the Group 11 and 12 metals. Metallothioneins are characterised by the presence of numerous cys-x-cys and cys-cys motifs in the sequence and are found naturally with either one domain or two, linked, metal-binding domains. The use of chains of these metal-thiolate domains offers the possibility of creating chemically tuneable and, therefore, chemically dependent electrochemical or photochemical surface modifiers or as nanomachinery with nanomechanical properties. In this work, the metal-binding properties of the Cd(4)-containing domain of alpha-rhMT1a assembled into chains of two and three concatenated domains, that is, "necklaces", have been studied by spectrometric techniques, and the interactions within the structures modelled and interpreted by using molecular dynamics. These chains are metallated with 4, 8 or 12 Cd(II) ions to the 11, 22, and 33 cysteinyl sulfur atoms in the alpha-rhMT1a, alphaalpha-rhMT1a, and alphaalphaalpha-rhMT1a proteins, respectively. The effect of pH on the folding of each protein was studied by ESI-MS and optical spectroscopy. MM3/MD simulations were carried out over a period of up to 500 ps by using force-field parameters based on the reported structural data. These calculations provide novel information about the motion of the clustered metallated, partially demetallated, and metal-free peptide chains, with special interest in the region of the metal-binding site. The MD energy/time trajectory conformations show for the first time the flexibility of the metal-sulfur clusters and the bound amino acid chains. We report unexpected and very different sizes for the metallated and demetallated proteins from the combination of experimental data, with molecular dynamics simulations.

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Information on Metal Binding Properties of Metallothioneins from Optical Spectroscopy

Cited by 48 publications

References 8 publications

Copper Binding to Rabbit Liver Metallothionein

Copper Binding to Rabbit Liver Metallothionein

Metal Ion Trafficking in Earthworms

Metallobiological Necklaces: Mass Spectrometric and Molecular Modeling Study of Metallation in Concatenated Domains of Metallothionein

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