Eva Freisinger

University of Zurich, Zürich, ZH, Switzerland

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Publications (96)319.21 Total impact

  • Article: Preface.
    Eva Freisinger, Roland K O Sigel
    Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry. 08/2014; 19(Supplement 2):699.
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    ABSTRACT: Getting to the heart of it: Co-crystallization of an RNA three-way junction with a cylindrical di-iron(II)-based anti-cancer drug (green) results in π-stacking interactions between the cylinder and the central base pairs of the RNA structure. The shape, size, and cationic nature of the cylinder were found to be responsible for this perfect fit. Native gel electrophoresis studies confirmed stabilization of the RNA three-way junction by the iron(II) cylinder.
    Angewandte Chemie International Edition 08/2013; · 11.34 Impact Factor
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    ABSTRACT: Metallothioneins (MTs) are among others involved in the cellular regulation of essential Zn(II) and Cu(I) ions. However, the high binding affinity of these proteins requires additional factors to promote metal ion release under physiological conditions. The mechanisms and efficiencies of these processes leave many open questions. We report here a comprehensive analysis of the Zn(II)-release properties of various MTs with special focus on members of the four main subfamilies of plant MTs. Zn(II) competition experiments with the metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) in the presence of the cellular redox pair glutathione (GSH)/glutathione disulfide (GSSG) show that plant MTs from the subfamilies MT1, MT2, and MT3 are remarkably more affected by oxidative stress than those from the Ec subfamily and the well-characterized human MT2 form. In addition, we evaluated proteolytic digestion with trypsin and proteinase K as an alternative mechanism for selective promotion of metal ion release from MTs. Also here the observed percentage of liberated metal ions depends strongly on the MT form evaluated. Closer evaluation of the data additionally allowed deducing the thermodynamic and kinetic properties of the Zn(II) release processes. The Cu(I)-form of chickpea MT2 was used to exemplify that both oxidation and proteolysis are also effective ways to increase the transfer of copper ions to other molecules. Zn(II) release experiments with the individual metal-binding domains of Ec-1 from wheat grain reveal distinct differences from the full-length protein. This triggers the question about the roles of the long cysteine-free peptide stretches typical for plant MTs.
    Metallomics 07/2013; · 4.10 Impact Factor
  • Xiaoqiong Wan, Oliver Schicht, Eva Freisinger
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    ABSTRACT: The plant metallothioneins (MTs) show high sequence diversity. Proposed to function mainly in metal ion homeostasis and detoxification these small cysteine-rich proteins exhibit pronounced affinities to metal ions with the electron configuration d10. Having previously studied the coordination abilities of two MTs from Cicerarietinum (chickpea) for divalent metal ions we now aim to expand the knowledge to the binding characteristics for CuI. Performing titration studies followed by UV and circular dichroism spectroscopy distinct differences between the two proteins are revealed.
    Zeitschrift für anorganische Chemie 07/2013; 639(8‐9). · 1.16 Impact Factor
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    ABSTRACT: A spring from a former copper shale mine in the area of Mansfelder Land, Germany, shows extremely high transition metal ion concentrations, i.e. 40mM Zn(II), 208μM Cu(II), 61μM As(V), and 25μM Cd(II). This makes it a challenging habitat for living organisms as they have to cope with metal ion concentrations that by far exceed the values usually observed in spring water. One of the surviving species found is the aquatic fungus Heliscus lugdunensis (teleomorph: Nectria lugdunensis). Investigation of its redox related heavy metal tolerance revealed the presence of small thiol containing compounds as well as a small metallothionein, Neclu_MT1 (MT1_NECLU: P84865). While Cd(II)-induction of metallothioneins is observed in many species, the fact that exclusively Cd(II), but not Zn(II), Cu(I), As(III) or oxidative stress can induce Neclu_MT1 protein synthesis is unparalleled. To complement the physiological studies performed in the fungus H. lugdunensis, the Cd(II) and Zn(II) binding characteristics of the recombinantly expressed protein were spectroscopically analysed in vitro aiming to demonstrate the observed Cd(II) specificity also on the protein level. Stoichiometric analyses of the recombinant protein in combination with photospectrometric metal ion titrations and (113)Cd-NMR experiments reveal that metal ion binding capacities and consequently the structures formed at physiological Neclu_MT1 concentrations differ from each other. Concluding, we describe the first solely Cd(II)-inducible metallothionein, Neclu_MT1, from H. lugdunensis, featuring a difference in the structure of the Cd(II)versus the Zn(II) metalated protein in a physiologically relevant concentration range.
    Journal of inorganic biochemistry 06/2013; · 3.25 Impact Factor
  • Tamara Huber, Eva Freisinger
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    ABSTRACT: Metallothioneins are small cysteine-rich proteins coordinating various transition metal ions preferably with the electron configuration d(10). They are ubiquitously present in all phyla, and next to phytochelatins they represent a successful molecular concept for high-capacity metal ion binding. Recent studies showed the incorporation of sulfide ions into the metal-thiolate cluster of metallothionein 2 from the plant Cicer arietinum (cicMT2) increasing the cadmium binding capacity and stability of the cluster. In the present work, the sulfide-induced structural changes accompanying the cluster formation and the sulfide-modulated increase in cluster size are analyzed in detail with a variety of analytical and spectroscopic techniques. Evaluation of the mechanism of sulfide containing Cd(II)-thiolate cluster formation in cicMT2 reveals a strong dependence on the sequence of metal and sulfide additions for successful sulfide incorporation. To probe the general ability of metallothioneins to form sulfide containing larger metal-thiolate clusters, analogous experiments were performed with a mammalian metallothionein. The observation that the cadmium binding ability of rabbit liver MT2A was only slightly increased led to the development of a hypothesis in which the long cysteine-free linker regions present in certain plant metallothioneins may contribute to the accommodation of the respective larger cluster assemblies.
    Dalton Transactions 05/2013; · 3.81 Impact Factor
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    ABSTRACT: The first cyclic analog of a metallothionein (MT) was prepared and analyzed by UV and (magnetic) circular dichroism spectroscopy, ESI-MS as well as NMR spectroscopy. Results reveal that the evaluated cyclic g-Ec-1 domain of the wheat MT Ec-1 retains its ability to coordinate two Zn(II) or Cd(II) ions and adopts a three-dimensional structure that is highly similar to the one of the linear wild-type form. However, the reduced flexibility of the protein backbone facilitates structure solution significantly and results in a certain stabilization of metal binding to the protein.
    Molecules 01/2013; 18(11):14414-14429. · 2.43 Impact Factor
  • Eva Freisinger, Milan Vašák
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    ABSTRACT: Metallothioneins (MTs) are low-molecular-mass cysteine-rich proteins with the ability to bind mono- and divalent metal ions with the electron configuration d ( 10 ) in form of metal-thiolate clusters. MTs are thought, among others, to play a role in the homeostasis of essential Zn(II) and Cu(I) ions. Besides these metal ions also Cd(II) can be bound to certain MTs in vivo, giving rise to the perception that another physiological role of MTs is in the detoxification of heavy metal ions. Substitution of the spectroscopically silent Zn(II) ions in metalloproteins by Cd(II) proved to be an indispensable tool to probe the Zn(II) sites in vitro. In this review, methods applied in the studies of structural and chemical properties of Cd-MTs are presented. The first section focuses on the physical basis of spectroscopic techniques such as electronic absorption, circular dichroism (CD), magnetic CD, X-ray absorption, and perturbed angular correlation of γ-rays spectroscopy, as well as mass spectrometry, and their applications to Cd-MTs from different organisms. The following is devoted to the discussion of metal binding affinities of Cd-MTs, cluster dynamics, the reactivity of bound Cd(II) ions with metal ion chelators and of thiolate ligands with alkylating and oxidizing agents. Finally, a brief summary of the known three-dimensional structures of Cd-MTs, determined almost exclusively by multinuclear NMR techniques, is presented. Besides Cd-MTs, the described methods can also be applied to the study of metal binding sites in other metalloproteins.
    Metal ions in life sciences. 01/2013; 11:339-71.
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    ABSTRACT: A new family of hydrazone-based nucleosides for use in metal-mediated base pairs was devised. The artificial nucleobases are derivatives of the papy ligand (papy = pyridinecarboxaldehyde-2′-pyridylhydrazone). By replacing the pendant pyridine moiety in papy by furan and thiophene, respectively, tridentate nucleosides with N, N, N-, N, N, O- and N, N, S-donor sites were obtained. As only a few transition metal complexes with pendant furan ligands have been reported, a model nucleobase for the N, N, O-donor nucleoside was synthesized. The molecular structures of its Cu2+, Ni2+, and Co2+ complexes are reported. In all complexes, only weak M–O(furan) bonding is observed. The Co2+ complex displays a pentagonal bipyramidal coordination arrangement. In general, the structures of the metal complexes suggest that the respective nucleosides can be applied in metal-mediated base pairs.
    Zeitschrift für anorganische Chemie 01/2013; · 1.16 Impact Factor
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    ABSTRACT: Recognition and stabilization of an RNA three‐way junction by a supramolecular di‐iron(II) cylindrical complex is described by R. K. O. Sigel, B. Spingler, M. J. Hannon, E. Freisinger et al. in their Communication on page 11513 ff. This potential anti‐cancer metal‐based drug fits perfectly into the central RNA core and stabilizes this architecture in the solid state as well as under native gel conditions. (Cover picture: Joachim Schnabl.)
    Angewandte Chemie International Edition 01/2013; 52(44). · 11.34 Impact Factor
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    ABSTRACT: Erkennung und Stabilisierung einer Y‐verzweigten RNA durch einen zylinderförmigen supramolekularen Dieisen(II)‐Komplex werden von R. K. O. Sigel, B. Spingler, M. J. Hannon, E. Freisinger et al. in der Zuschrift auf S. 11727 ff. beschrieben. Der potenzielle Antikrebswirkstoff passt genau in den zentralen RNA‐Hohlraum und stabilisiert die Architektur im Festkörper ebenso wie unter nativen Gelbedingungen. (Bild: Joachim Schnabl)
    Angewandte Chemie 01/2013; 125(44).
  • Xiaoqiong Wan, Eva Freisinger
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    ABSTRACT: The plant metallothionein2 from Cicer arietinum (chickpea), cic-MT2, is known to coordinate five divalent metal ions such as Zn(II) or Cd(II), which are arranged in a single metal thiolate cluster. When the Zn(II) form of the protein is titrated with Cd(II) ions in the presence of sulfide ions, an increased Cd(II) binding capacity and concomitant incorporation of sulfide ions into the cluster are observed. The exact stoichiometry of this novel cluster, its spectroscopic properties, and the significantly increased pH stability are analyzed with different techniques, including UV and circular dichroism spectroscopy and colorimetric assays. Limited proteolytic digestion provides information about the spacial arrangement of the cluster within the protein. Increasing the Cd(II) scavenging properties of a metallothionein by additionally recruiting sulfide ions might be an economic and very efficient detoxification strategy for plants.
    Inorganic Chemistry 12/2012; · 4.59 Impact Factor
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    ABSTRACT: Metal-mediated base pairs can be used to insert metal ions into nucleic acids at precisely defined positions. As structural data on the resulting metal-modified DNA are scarce, appropriate model complexes need to be synthesized and structurally characterized. Accordingly, the molecular structures of nine transition metal complexes of N-methyl-2, 2'-dipicolylamine (dipic) are reported. In combination with an azole-containing artificial nucleoside, this tridentate ligand had recently been used to generate metal-mediated base pairs (Chem. Commun. 2011, 47, 11041–11043). The PdII and PtII complexes reported here confirm that the formation of planar complexes (as required for a metal-mediated base pair) comprising N-methyl-2, 2'-dipicolylamine is possible. Two HgII complexes with differing stoichiometry indicate that a planar structure might also be formed with this metal ion, even though it is not favored. In the complex [Ag2(dipic)2](ClO4)2, the two AgI ions are located close to one another with an Ag···Ag distance of 2.9152(3) Å, suggesting the presence of a strong argentophilic interaction.
    Zeitschrift für anorganische Chemie 09/2012; 638(11). · 1.16 Impact Factor
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    ABSTRACT: Multinuclear and multidimensional nuclear magnetic resonance (NMR) spectroscopy is applied in our groups to gain insights into the role of metal ions for the function and structure of large biomolecules. Specifically, NMR is used i) to investigate how metal ions bind to nucleic acids and thereby control the folding and structure of RNAs, ii) to characterize how metal ions are able to stabilize modified nucleic acids to be used as potential nanowires, and iii) to characterize the formation, structure, and role of the diverse metal clusters within plant metallothioneins. In this review we summarize the various NMR experiments applied and the information obtained, demonstrating the important and fascinating part NMR spectroscopy plays in the field of bioinorganic chemistry.
    CHIMIA International Journal for Chemistry 01/2012; 66(10):791-7. · 1.09 Impact Factor
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    ABSTRACT: 2,2'-Bipyrazine (2,2'-bpz) reacts with cis-(NH(3))(2)Pt(II) in water to give a variety of products, several of which were isolated and characterized by X-ray analysis: cis-[Pt(NH(3))(2)(2,2'-bpz-N4)(2)](NO(3))(2)·3H(2)O (1), [{cis-Pt(NH(3))(2)(2,2'-bpz-N4,N4')}(3)]-(PF(6))(5)NO(3)·7H(2)O (2a), [{cis-Pt(NH(3))(2)(2,2'-bpz-N4,N4')}(3)](BF(4))(2)-(SiF(6))(2)·15H(2)O (2b), and [{cis-Pt(NH(3))(2)(2,2'-bpz-N4,N4')}(4)]-(SO(4))(4)·22H(2)O (3). In 1, 2b, and 3 the 2,2'-bpz ligands adopt approximately C(2h) symmetries, hence the two pyrazine halves are in trans orientation, whereas in 2a all three 2,2'-bpz bridges are approximately C(2v) symmetric, with the pyrazine halves cis to each other. The topologies of the two triangular complexes 2a and 2b are consequently distinctly different, but nevertheless both cations act as hosts for anions. In 2a a PF(6)(-) and a NO(3)(-) anion are associated simultaneously with the +6 cation, whereas in 2b it is a BF(4)(-) anion and a water molecule, which are trapped in its cavity. There is no anion inclusion in case of the metallasquare 3. In principle, 3 can exist in a large number of stereoisomers, depending on the rotational states of the bridging 2,2'-bpz ligands. Isolation of a single rotamer form of 3 with C(2h) symmetric 2,2'-bpz ligands and an overall meso form is proposed to be a consequence of a highly efficient self-assembly process that starts from the precursor 1 and reaction with two cis-(NH(3))(2)Pt(II) units. This process leads to the isolated rotamer of 3 regardless of whether two cations 1 in head-head form react with two cis-(NH(3))(2)Pt(II), or whether the Δ enantiomer of the chiral head-tail form of 1 combines with its Λ enantiomer through two cis-(NH(3))(2)Pt(II) entities.
    Chemistry 08/2011; 17(38):10771-80. · 5.93 Impact Factor
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    ABSTRACT: The novel C2h-symmetrical tetranuclear metallacycles (III) and (IV) crystallize isotypically in the triclinic space group P with Z = 1 (single crystal XRD).
    ChemInform 06/2011; 42(25).
  • Eva Freisinger
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    ABSTRACT: The metallothionein (MT) superfamily combines a large variety of small cysteine-rich proteins from nearly all phyla of life that have the ability to coordinate various transition metal ions, including Zn(II), Cd(II), and Cu(I). The members of the plant MT family are characterized by great sequence diversity, requiring further subdivision into four subfamilies. Very peculiar and not well understood is the presence of rather long cysteine-free amino acid linkers between the cysteine-rich regions. In light of the distinct differences in sequence to MTs from other families, it seems obvious to assume that these differences will also be manifested on the structural level. This was already impressively demonstrated with the elucidation of the three-dimensional structure of the wheat E(c)-1 MT, which revealed two metal cluster arrangements previously unprecedented for any MT. However, as this structure is so far the only one available for the plant MT family, other sources of information are in high demand. In this review the focus is thus set on any structural features known, deduced, or assumed for the plant MT proteins. This includes the determination of secondary structural elements by circular dichroism, IR, and Raman spectroscopy, the analysis of the influence of the long linker regions, and the evaluation of the spatial arrangement of the sequence separated cysteine-rich regions with the aid of, e.g., limited proteolytic digestion. In addition, special attention is paid to the contents of divalent metal ions as the metal ion to cysteine ratios are important for predicting and understanding possible metal-thiolate cluster structures.
    European Journal of Biochemistry 06/2011; 16(7):1035-45. · 3.42 Impact Factor
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    ABSTRACT: C2h-symmetrical tetranuclear metallacycles {[M(en)(CN)]4}(NO3)4 with M = PdII (4) and PtII (5) have been prepared upon reacting M(en)(CN)2 [M = PdII (1), PtII (2)] with [M(en)(H2O)2](NO3)2. Replacement of the nitrate anions of 5 by terephthalate anions yields the corresponding salt 5a. The X-ray crystal structures of 1, 4, 5, and 5a have been determined. In the metallacycles 4, 5, and 5a the four metals form almost ideal squares with average M···M distances of ca. 5.05 Å (5, 5a) and 5.08 Å (4) along the sides. As shown by 1H NMR spectroscopy, the Pt square 5 is stable in aqueous solution, whereas the Pd square 4 undergoes rearrangement reactions upon aging or the presence of other Pd species such as (bpy)PdII. Preliminary studies on the possibility of non-covalent interactions of 4 and 5 with model nucleobases in water reveal that only 5 is useful in this respect. According to the concentration-dependence 1H NMR study, there is an interaction with the purine base 9-ethyladenine, molecular details of which are unclear at this stage, however. Compound 4 is substitutionally labile and is transformed into the coordination compound 8 with 1-methylcytosine. Two more side products, produced during the various reactions carried out, were characterized by X-ray crystallography: [Pt(en)2][Pt(CN)4] (3) and [Pd(bpy)(en)](SO4)·3H2O (7).
    Berichte der deutschen chemischen Gesellschaft 04/2011; 2011(10). · 2.94 Impact Factor
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    ABSTRACT: Metallothioneins (MTs) are small cysteine-rich proteins coordinating various transition metal ions, including Zn(II), Cd(II), and Cu(I). MTs are ubiquitously present in all phyla, indicating a successful molecular concept for metal ion binding in all organisms. The plant MT E(c)-1 from Triticum aestivum, common bread wheat, is a Zn(II)-binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding β(E) domain was recently described. Here we present the structure of the N-terminal second domain, γ-E(c)-1, determined by NMR spectroscopy. The γ-E(c)-1 domain enfolds an M (2) (II) Cys(6) cluster and was characterized as part of the full-length Zn(6)E(c)-1 protein as well as in the form of the separately expressed domain, both in the Zn(II)-containing isoform and the Cd(II)-containing isoform. Extended X-ray absorption fine structure analysis of Zn(2)γ-E(c)-1 clearly shows the presence of a ZnS(4) coordination sphere with average Zn-S distances of 2.33 Å. (113)Cd NMR experiments were used to identify the M(II)-Cys connectivity pattern, and revealed two putative metal cluster conformations. In addition, the general metal ion coordination abilities of γ-E(c)-1 were probed with Cd(II) binding experiments as well as by pH titrations of the Zn(II) and Cd(II) forms, the latter suggesting an interaction of the γ domain and the β(E) domain within the full-length protein.
    European Journal of Biochemistry 03/2011; 16(5):683-94. · 3.42 Impact Factor
  • Eva Freisinger
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    ABSTRACT: While structures of mammalian metallothioneins and their metal-thiolate clusters have been known for nearly 20 years, the first three-dimensional structure of a plant metallothionein was only published very recently. Also in other aspects, research on plant metallothioneins lags behind what is known about the vertebrate forms. However, in the past years more and more information, mostly based on experiments performed with optical spectroscopy has become available and permit sometimes unforeseen insights. In the following, the different experimental strategies applied in our laboratory will be described together with the progress we have made over the past years in detecting and characterizing the metal-thiolate clusters of plant metallothioneins.
    CHIMIA International Journal for Chemistry 01/2010; 64(4):217-24. · 1.09 Impact Factor

Publication Stats

497 Citations
319.21 Total Impact Points

Institutions

  • 2007–2013
    • University of Zurich
      • Institut für Anorganische Chemie
      Zürich, ZH, Switzerland
  • 1997–2011
    • Technische Universität Dortmund
      • Faculty of Chemistry
      Dortmund, North Rhine-Westphalia, Germany
  • 2003
    • Stony Brook University
      • Center for Structural Biology
      Stony Brook, NY, United States
  • 2002
    • University of California, Berkeley
      Berkeley, California, United States
  • 1998
    • University of Queensland
      Brisbane, Queensland, Australia
    • University of Virginia
      Charlottesville, Virginia, United States