Eva Freisinger

University of Zurich, Zürich, Zurich, Switzerland

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Publications (104)422.85 Total impact

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    ABSTRACT: Mono(nucleobase) complexes of the general composition cis-[PtCl2 (NH3 )L] with L=1-methylcytosine, 1-MeC (1 a) and L=1-ethyl-5-methylcytosine, as well as trans-[PtX2 (NH3 )(1-MeC)] with X=I (5 a) and X=Br (5 b) have been isolated and were characterized by X-ray crystallography. The Pt coordination occurs through the N3 atom of the cytosine in all cases. The diaqua complexes of compounds 1 a and 5 a, cis-[Pt(H2 O)2 (NH3 )(1-MeC)](2+) and trans-[Pt(H2 O)2 (NH3 )(1-MeC)](2+) , display a rich chemistry in aqueous solution, which is dominated by extensive condensation reactions leading to μ-OH- and μ-(1-MeC(-) -N3,N4)-bridged species and ready oxidation of Pt to mixed-valence state complexes as well as diplatinum(III) compounds, one of which was characterized by X-ray crystallography: h,t-[{Pt(NH3 )2 (OH)(1-MeC(-) -N3,N4)}2 ](NO3 )2 ⋅2 [NH4 ](NO3 )⋅2 H2 O. A combination of (1) H NMR spectroscopy and ESI mass spectrometry was applied to identify some of the various species present in solution and the gas phase, respectively. As it turned out, mass spectrometry did not permit an unambiguous assignment of the structures of +1 cations due to the possibilities of realizing multiple bridging patterns in isomeric species, the occurrence of different tautomers, and uncertainties regarding the Pt oxidation states. Additionally, compound 1 a was found to have selective and moderate antiproliferative activity for a human cervix cancer line (SISO) compared to six other human cancer cell lines.
    No preview · Article · Oct 2015 · Chemistry - A European Journal
  • Katsiaryna Tarasava · Eva Freisinger
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    ABSTRACT: While Zn(II) and Cd(II) have similar geochemical and environmental properties, their biological properties are distinctively different as Cd(II) ions have very limited metabolic significance and are mostly even toxic, while Zn(II) ions belong to the most essential micronutrients. One of the key proteins involved in intracellular Zn(II) and Cd(II) binding are metallothioneins (MTs), small cysteine-rich proteins ubiquitously found in many different organisms. In the past two decades, also MT sequences from diverse species that contain histidine residues have been found, and His-metal ion coordination has been shown. It is not clear, however, why in some MTs parts of the Cys residues are replaced by His, while most other MTs only contain Cys residues for metal ion binding. To address this question, we used the γ-domain of the early-cysteine labeled (Ec-1) metallothionein from common wheat as a model system because its enclosed M2Cys6 cluster represents the smallest metal-thiolate cluster possible with divalent metal ions. Based on the known three-dimensional structure of the γ-domain we set about to investigate the influence of a single Cys-to-His mutation on the structure and metal ion binding abilities of this domain. Combined data obtained by mass spectrometry, UV, as well as NMR spectroscopy suggest a preference for Zn(II) versus Cd(II) ions in the histidine containing binding site. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Aug 2015 · Journal of inorganic biochemistry
  • Eva Freisinger · Roland Sigel

    No preview · Article · Jul 2015 · Journal of inorganic biochemistry
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    ABSTRACT: Potentiometric pH titrations and pD dependent (1)H NMR spectroscopy have been applied to study the acidification of the exocyclic amino group of adenine (A) model nucleobases (N9 position blocked by alkyl groups) when carrying trans-a2Pt(II) (with a=NH3 or CH3NH2) entities both at N1 and N7 positions. As demonstrated, in trinuclear complexes containing central A-Pt-A units, it depends on the connectivity pattern of the adenine bases (N7/N7 or N1/N1) and their rotamer states (head-head or head-tail), how large the acidifying effect is. Specifically, a series of trinuclear complexes with (A-N7)-Pt-(N7-A) and (A-N1)-Pt-(N1-A) cross-linking patterns and terminal 9-alkylguanine ligands (9MeGH, 9EtGH) have been analyzed in this respect, and it is shown that, for example, the 9MeA ligands in trans-,trans-,trans-[Pt(NH3)2(N7-9MeA-N1)2{Pt(NH3)2(9EtGH-N7)}2](ClO4)6·6H2O (4a) and trans-,trans-,trans-[Pt(NH3)2(N7-9EtA-N1)2{Pt(CH3NH2)2(9-MeGH-N7)}2](ClO4)6·3H2O (4b) are more acidic, by ca. 1.3units (first pKa), than the linkage isomer trans-,trans-,trans-[Pt(CH3NH2)2(N1-9MeA-N7)2{Pt(NH3)2(9MeGH-N7)}2](NO3)6·6.25H2O (1b). Overall, acidifications in these types of complexes amount to 7-9units, bringing the pKa values of such adenine ligands in the best case close to the physiological pH range. Comparison with pKa values of related trinuclear Pt(II) complexes having different co-ligands at the Pt ions, confirms this picture and supports our earlier proposal that the close proximity of the exocyclic amino groups in a head-head arrangement of (A-N7)-Pt-(N7-A), and the stabilization of the resulting N6H(-)⋯H2N6 unit, is key to this difference. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Feb 2015 · Journal of Inorganic Biochemistry
  • David Egloff · Igor A Oleinich · Eva Freisinger
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    ABSTRACT: DNA lesions such as 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC) are ubiquitously present in genomes of different organisms and show increasing levels upon exposure to mutagenic substances or under conditions of chronic inflammations and infections. To facilitate investigations of the mutagenic properties and repair mechanisms of etheno-base adducts, access to oligonucleotides bearing these lesions at defined positions is of great advantage. In this study we report a new synthetic strategy to sequence-specifically generate etheno-adducts in a single-stranded unmodified DNA sequence making use of a DNA-templated approach that positions the alkylating agent close in space to the respective target base. In contrast to solid-phase synthesis of modified oligonucleotides such DNA-templated methods can be applied to single-stranded nucleic acids of unrestricted lengths. The modular nature of the system allows straightforward adaptation to different sequences.
    No preview · Article · Nov 2014 · ACS Chemical Biology
  • Katsiaryna Tarasava · Eva Freisinger
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    ABSTRACT: Head-to-tail backbone cyclization of proteins is a widely used approach for the improvement of protein stability. One way to obtain cyclic proteins via recombinant expression makes use of engineered Intein tags, which are self-cleaving protein domains. In this approach, pH-induced self-cleavage of the N-terminal Intein tag generates an N-terminal cysteine residue at the target protein, which then attacks in an intramolecular reaction the C-terminal thioester formed by the second C-terminal Intein tag resulting in the release of the cyclic target protein. In the current work we aimed to produce a cyclic analog of the small γ-Ec-1 domain of the wheat metallothionein, which contains six cysteine residues. During the purification process we faced several challenges, among them premature cleavage of one or the other Intein tag resulting in decreasing yields and contamination with linear species. To improve efficiency of the system we applied a number of optimizations such as the introduction of a Tobacco etch virus cleavage site and an additional poly-histidine tag. Our efforts resulted in the production of a cyclic protein in moderate yields without any contamination with linear protein species.
    No preview · Article · Oct 2014 · Protein Engineering Design and Selection
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    Article: Preface.
    Eva Freisinger · Roland K O Sigel

    Preview · Article · Aug 2014
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    Katsiaryna Tarasava · Silke Johannsen · Eva Freisinger
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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.
    Preview · Article · Nov 2013 · Molecules
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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.
    No preview · Article · Oct 2013 · Angewandte Chemie International Edition
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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)
    Full-text · Article · Oct 2013 · Angewandte Chemie
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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.)
    No preview · Article · Oct 2013 · Angewandte Chemie International Edition
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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.
    Preview · Article · Jul 2013 · Metallomics
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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.
    Full-text · Article · Jul 2013 · Zeitschrift für anorganische Chemie
  • 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.
    No preview · Article · Jul 2013 · Zeitschrift für anorganische Chemie
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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.
    Full-text · Article · Jun 2013 · Journal of inorganic biochemistry
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    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.
    Preview · Article · May 2013 · Dalton Transactions
  • 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.
    No preview · Article · Feb 2013 · Metal ions in life sciences
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    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.
    Preview · Article · Dec 2012 · Inorganic Chemistry
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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.
    Preview · Article · Oct 2012 · CHIMIA International Journal for Chemistry
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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.
    No preview · Article · Sep 2012 · Zeitschrift für anorganische Chemie

Publication Stats

2k Citations
422.85 Total Impact Points


  • 2004-2015
    • University of Zurich
      • Institut für Anorganische Chemie
      Zürich, Zurich, Switzerland
  • 1997-2011
    • Technische Universität Dortmund
      • Faculty of Chemistry
      Dortmund, North Rhine-Westphalia, Germany
    • University of Helsinki
      • Department of Chemistry
      Helsinki, Uusimaa, Finland
  • 2003
    • New York Structural Biology Center
      New York City, New York, United States
  • 2002
    • Stony Brook University
      • Center for Structural Biology
      Stony Brook, New York, United States
  • 1998
    • Università degli Studi di Trieste
      Trst, Friuli Venezia Giulia, Italy