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Metal-Metallothionein complexes: insights into structural features by Raman spectroscopy.
No full textMetallothioneins (MTs) are low molecular weight, cysteine-rich proteins with an exceptional heavy metal coordination capacity. Because of their ability to bind metals and to scavenge oxidant radicals, MTs are considered to play a role in metal homeostasis, metal detoxificalion and control of the oxidative stress. Although their high heterogeneity on the expression patterns, metal binding abilities and primary structure suggest very diverse functional specializations, the structural and functional studies have been mainly devoted to vertebrate and fungal MTs. Participation of metal ligands other than the cysteines and the presence of secondary structure elements in metal-MT complexes are fairly unknown, especially in non-vertebrate MTs. Recently, it has been shown that ligands other than Cys can participate in the coordination sphere of metals in MTs. Two main types have been identified: endogenous ligands such as imidazole moiety of His residues and /or exogenous ligands such as inorganic ions (i.e. sulfide or chloride ions)
Structural study of the zinc and cadmium complexes of a type 2 plant (Quercus suber) metallothionein: insights by vibrational spectroscopy.
No full textZn- and Cd-complexes of Quercus suber metallothionein (QsMT) were obtained by in vivo-synthesis, in order to obtain physiologically representative aggregates, and characterized by spectrometric and spectroscopic methods. The secondary structure elements and the coordination environments of the metal binding sites of the two aggregates were determined, as well as the main metal-containing species formed. The results obtained from the analysis of the Raman and IR spectra reveal that these metal-MT complexes predominantly contain beta-sheet elements (about 60%), whereas they lack alpha-helices. These structural features slightly depend on the divalent metal bound. In particular, Cd(II) binding to QsMT induces a slight increase of the beta-sheet percentage, as well as a decrease in beta-turn elements with respect to Zn(II) binding. Conversely, the in vivo capability of QsMT to inglobe metal and sulfide ions is metal-depending. Spectroscopic vibrational data also confirm the presence of sulfide ligands in the metal clusters of both Zn- and Cd-QsMT, while the participation of the spacer His residue in metal coordination was only found in Cd-QsMT, in agreement with the CD results. Overall data suggest different coordination environments for Zn(II) and Cd(II) ions in QsMT. Copyright 2007 Wiley Periodicals, Inc
Raman study of in vivo synthesized Zn(II)-metallothionein complexes: structural insight into metal clusters and protein folding.
No full textMetallothioneins (MTs) are metal-chelating peptides that play an active role in zinc homeostasis. The participation of metal ligands other than cysteines and the presence of secondary structure elements in metal-MT complexes are fairly unknown, especially in nonvertebrate MTs. Here, four Zn(II) complexes of invertebrate MTs (mollusc, insect, nematode, and echinoderm) and the Zn(II)-MT complex of the mammalian MT1 isoform, heterologously synthesized in E. coli, were studied by analytic and spectroscopic techniques. By Raman and circular dichroism spectroscopy, new structural informations were obtained. The five analyzed MT isoforms consist largely of -turns with the near exclusion of -helical segments. Raman spectroscopy was revealed as an useful tool, providing information about the state of the cysteine sulfur atoms (metal coordinated and oxidized), the participation of histidine in metal coordination, and the molecular environment of tyrosine residues. In all the five Zn(II)-MT studied samples, acid-labile sulfide anions were found as nonproteic ligands, since sulfide-containing and sulfide-devoid species coexisted in the corresponding preparations. Significantly, Raman bands useful as markers of sulfide bridging ligands were identified. Overall, this work illustrates how the combination of analytical and spectroscopic techniques can be a very informative approach for the analysis of in vivo-synthesized metal-MT complexes, providing new data on the metal binding behavior of MTs from the most diverse organisms. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 1114-1124, 2008.
This article was originally published online as an accepted preprint. The Published Online date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [email protected]
Raman spectroscopy investigation on metal-metallothionein complexes.
No full textMetallothioneins (MTs) are low molecular weight, cysteine-rich proteins with an exceptional heavy metal coordination capacity. Because of their ability to bind metals and to scavenge oxidant radicals, MTs are considered to play a role in metal homeostasis, metal detoxification and control of the oxidative stress. Their high heterogeneity, metal binding abilities and primary structure suggest very diverse functional specializations. Structural and functional studies have been mainly devoted to vertebrate MTs. Participation of metal ligands other than Cys and the presence of secondary structure elements in metal-MT complexes are fairly unknown, especially in non-vertebrate MTs. Recently, it has been shown that other ligands can participate in metal coordination; two main ligand types have been identified: endogenous, as His residues and exogenous, as inorganic ions (i.e. S2- or Cl-).
In vivo-synthesized Zn-MTs, representative of different families (mollusc, insect, nematode, echinoderm, vertebrate and plant, enclosing the mammalian MT1 isoform), were heterologously synthesized in E.coli and studied by analytic and spectroscopic techniques. The examined MTs (CeMT2, MeMT, SpMTA, MtnB, QsMT and MT1) contain 43-73 a.a., among which at least a 30% are Cys. To evaluate the influence of the metal on the MT structure, also the Cd-QsMT was synthesised and analysed in comparison with Zn-QsMT. Almost all the MTs considered are devoid of aromatic residues (only Phe in SpMTA and QsMT, Tyr in CeMT2, and His in CeMT2 and QsMT) and contain variable amounts of metal and S2- ions, quantitatively evaluated by acid ICP-AES and GC-FPD measurements. The formation of more than one species (S2--containing and S2--devoid complexes), revealed by ESI-MS spectra, was evident for all the MTs.
The Raman spectra gave information about the structure of the metal-MT aggregates. In particular, the presence of secondary structure elements was determined: for the examined MTs isoforms, a relevant contribution of p-sheet and p-turns was shown, whereas the a-helix content resulted almost negligible. As regards Cys sulfurs, almost all Cys were involved in the metal coordination, as indicated by several bands attributable to metal-S stretching modes at low wavenumbers (<500 cm-1). In particular, the high number of vM-S bands, as well as their broadening, suggest the formation of different metal centres. Raman bands markers of sulfide bridging ligands were also identified. In Zn-
CeMT2 and Zn-QsMT the eventual participation of the His residue in metal binding was evaluated through a curve fitting analysis of thè 1630-1565 cm"' region. By considering the integrated intensity of the bands, it can be concluded that His residues are mainly coordinated in Zn-CeMT2 whereas in Zn-QsMT His is mainly present as free tautomer. The combination of analytical and spectroscopic techniques has been highly informative for the analysis of in vivo-synthesized metal-MT complexes; Raman studies revealed one of the most promising experimental strategies to provide new data on the knowledge of the metal binding behavior of MTs from the most diverse organisms
Comparative insight into the Zn(II)-, Cd(II)- and Cu(I)-binding features of the protozoan Tetrahymena pyriformis MT1 metallothionein.
No full textTetrahymena pyriformis MT1 (TpyMT1) is a model among ciliate metallothioneins (MTs). Here, we report on the analytic (ICP-AES, GC-FPD), spectroscopic (CD, UV–Vis, Raman) and spectrometric (ESI-MS) characterization of its recombinant Cd(II)-, Zn(II)- and Cu(I)-complexes, and of those formed during in vitro Zn/Cd and Zn/Cu replacement. In the presence of Cd(II), TpyMT1 renders a major Cd11–TpyMT1 species, which is also the final step reached in the in vitro Zn/Cd exchange process in Zn11–TpyMT1. Spectroscopic data supports a different folding of the isostoichiometric Cd11– and Zn11–TpyMT1 complexes. Unexpectedly, TpyMT1 biosynthesis in Zn(II)-rich cultures was sensitive to the aeration degree, so that high oxygenation rendered undermetalated, partially-oxidized, complexes (Zn9–TpyMT1). Biosynthesis in Cu(I)-rich media rendered extremely heterogeneous mixtures of CuxZny-species (x + y = 8–20), where the higher the aeration, the higher the Zn(II) content. The complexity of these samples was reproduced during the Zn/Cu replacement, as the number of generated species increased gradually with the addition of copper to Zn11–TpyMT1. According to our results, a clear preference of TpyMT1 for Cd(II) binding, rather than for Zn(II), and especially Cu(I) can be postulated. This character is totally consistent with the induction pattern of the TpyMT1 gene and the postulated role of TpyMT1 in Cd-detoxification
Effects of radical stress on metal complexes of a plant metallotionein: desulfurisation reactions associated with the formation of trans lipids in model membrane.
No full textThe effects of radical stress in the biological environment is a very active field of research connecting varies discipline in life science. Thus, a comprehensive vision of all possible reactive species is necessary for contributing to the solution of puzzling questions on free radicals. In this contest, damages to Zn(II) and Cd(II) complexes of a metallothionein from a plant (Quercus suber -QsMT), due to radical stress exposure, were investigated.
Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins with high capacity for binding both essential (ZnII and CuI) and xenobiotic (CdII and HgII) metal ions. Metal-MT coordination is mainly achieved by formation of metal-thiolate bonds. Also the contribution of non-proteic ligands, such as chloride and/or sulfide anions, has been recently reported. As almost all the plant MTs, QsMT presents a peculiar amino acid sequence organization consisting of two Cys-rich domains (8 and 6 Cys each), linked by a cysteine-devoid spacer region which includes additional putative ligand (His).
Although MTs do not appear to be essential for life, there is mounting evidences for a survival advantage of MT in situations of stress, including exposure to radicals and toxic metals. In support to a MT function in control of oxidative stress, many studies have showed that MT expression increases most dramatically in response to tissue injury, inflammation, tumour and it appears to reduce apoptosis.
The reactions of reductive reactive species (H* atoms and eaq), produced by gamma-irradiation of water, with Zn- and Cd-QsMT were carried out in both aqueous solutions and vesicle suspensions, and were characterized by different approaches.
Using a biomimetic model based on unsaturated lipid vesicle suspensions the occurrence of tandem protein/lipid damage was shown The reactions of reductive reactive species with methionine residues and/or sulfur-containing ligands afford diffusible sulfur-centered radicals, which migrate from the aqueous phase to the lipid bilayer and transform the cis double bond of the oleate moiety to the trans isomer. Tailored experiments allowed the reaction mechanism to be defined in some details. The formation of sulfur-centered radicals is accompanied by the modification of the metal-QsMT complexes, which were monitored by various spectroscopic and spectrometric techniques (Raman and ESI-MS). The H* atoms and eaq attacks on the metal-QsMT aggregates are able to induce significant structural changes such as partial deconstruction and/or rearrangement of the metal clusters, and breaking of the protein backbone. In particular, Cys is among the most sensitive residues towards radical attack, suggesting that the thiolate clusters of both metal-QsMTs act as efficient interceptors of reducing species. The radical-induced effects are dependent on the divalent metal bound
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Effects of radical stress on metal complexes of a plant metallothionein: desulfurisation reactions associated with the formation of trans lipids in model membrane.
No full textThe effects of radical stress in the biological environment is a very active field of research connecting varies discipline in life science. Thus, a comprehensive vision of all possible reactive species is necessary for contributing to the solution of puzzling questions on free radicals. In this contest, damages to Zn(II) and Cd(II) complexes of a metallothionein from a plant (Quercus suber -QsMT), due to radical stress exposure, were investigated.
Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins with high capacity for binding both essential (ZnII and CuI) and xenobiotic (CdII and HgII) metal ions. Metal-MT coordination is mainly achieved by formation of metal-thiolate bonds. Also the contribution of non-proteic ligands, such as chloride and/or sulfide anions, has been recently reported. As almost all the plant MTs, QsMT presents a peculiar amino acid sequence organization consisting of two Cys-rich domains (8 and 6 Cys each), linked by a cysteine-devoid spacer region which includes additional putative ligand (His).
Although MTs do not appear to be essential for life, there is mounting evidences for a survival advantage of MT in situations of stress, including exposure to radicals and toxic metals. In support to a MT function in control of oxidative stress, many studies have showed that MT expression increases most dramatically in response to tissue injury, inflammation, tumour and it appears to reduce apoptosis.
The reactions of reductive reactive species (H' atoms and eaq-), produced by gamma-irradiation of water, with Zn- and Cd-QsMT were carried out in both aqueous solutions and vesicle suspensions, and were characterized by different approaches.
Using a biomimetic model based on unsaturated lipid vesicle suspensions the occurrence of tandem protein/lipid damage was shown The reactions of reductive reactive species with methionine residues and/or sulfur-containing ligands afford diffusible sulfur-centered radicals, which migrate from the aqueous phase to the lipid bilayer and transform the cis double bond of the oleate moiety to the trans isomer. Tailored experiments allowed the reaction mechanism to be defined in some details. The formation of sulfur-centered radicals is accompanied by the modification of the metal-QsMT complexes, which were monitored by various spectroscopic and spectrometric techniques (Raman and ESI-MS). The H' atoms and eaq- attacks on the metal-QsMT aggregates are able to induce significant structural changes such as partial deconstruction and/or rearrangement of the metal clusters, and breaking of the protein backbone. In particular, Cys is among the most sensitive residues towards radical attack, suggesting that the thiolate clusters of both metal-QsMTs act as efficient interceptors of reducing species. The radical-induced effects are dependent on the divalent metal bound
Metal complexes of a plant metallothionein under radical stress: assessment of structural modifications and transfer of radical damage.
No full textDamages to Zn2+and Cd2+ complexes of a metallothionein (MT) from a plant (Quercus suber - Qs), due to radicai stress exposure, were investigated. QsMT, obtained by in vivo synthesis, is a low-molecular weight cysteine-rich protein with high capacity for binding metal ions. Although MTs do not appear to be essential for life, there is mounting evidences for a survival advantage of MT in situations of stress, including exposure to radicals and toxic metals. Gamma-irradiation was used to simulate the conditions of an endogenous radical stress. The degradation of the metal complexes was followed by Raman spectroscopy and the occurrence of tandem protein/lipid damage was shown by using a biomimetic model based on unsaturated lipid vesicle suspensions. The H1 and eaq- attacks on the metal-QsMT aggregates are able to induce significant structural changes such as partial deconstruction and/or rearrangement of the metal clusters, and breaking of the protein backbone. Sulfur-containing residues resulted to be selectively attacked; in particular, Cys resulted to be among the most sensitive residues towards radical attack, suggesting that the thiolate clusters of both metal-QsMTs act as efficient interceptors of reducing species. Under reductive stress Zn-QsMT undergoes a significant thiolate group oxidation. The participation of His to metal coordination became necessary for protein stabilization after radical stress. The radical-induced effects were dependent on the divalent metal bound. The reactions of reductive reactive species with Met residues and/or sulfur-containing ligands afford diffusible sulfur-centered radicals, which migrate from the aqueous phase to the lipid bilayer and transform the cis doublé bond of thè oleate moiety to the trans isomer
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