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A. Structures and properties of plant metallothioneins and related artificial proteins - B. Site specific modifications of larger nucleic acids

English title A. Structures and properties of plant metallothioneins and related artificial proteins - B. Site specific modifications of larger nucleic acids
Applicant Freisinger Eva
Number 139194
Funding scheme SNSF Professorships
Research institution Institut für Chemie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Inorganic Chemistry
Start/End 01.06.2012 - 31.05.2014
Approved amount 797'309.00
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All Disciplines (4)

Discipline
Inorganic Chemistry
Biophysics
Biochemistry
Molecular Biology

Keywords (9)

plant metallothioneins; cyclic proteins; spectroscopy; X-ray crystallography; metal-thiolate clusters; bioinorganic chemistry; nucleic acids; site specific nucleobase modifications; DNA-templated synthesis

Lay Summary (English)

Lead
Lay summary

This scientific project is divided into two, largely independent subjects.

Part A focuses on the metal ion binding abilities of plant metallothioneins (MTs) and related artificial proteins, including the investigation of their structures and general properties. MTs are a superfamily of small cysteine-rich proteins which coordinate metal ions with the electron configuration d10, i.e. essential Zn(II) and Cu(I) as well as toxic Cd(II), Hg(II), As(III), Pb(II), etc. The metal ions are arranged in metal-thiolate cluster structures which greatly enhances the metal ion binding capacity of these proteins. They have a proposed role in metal homeostasis and detoxification, as well as in stress response, e.g. by scavenging reactive oxygen species. Compared to the vertebrate forms, plant MTs are considerably less well studied. They show pronounced sequence diversity and hence it can be expected that plant MTs combine a multitude of functions, properties and also structures. The latter we could already demonstrate in the previous project by determining the very first two structures of a plant MT ever by NMR spectroscopy, revealing unprecedented cluster structures. To increase the knowledge about plant MT properties and structures further, mainly spectroscopic methods including NMR spectroscopy for structural studies will be applied. The new knowledge gained might allow us to optimize the metal ion binding properties or even metal ion specificity of these proteins. Plants, over-expressing such specialized proteins, might be able to grow in polluted areas or might be used to specifically remove heavy metal ions from the environment (phytoremidation), e.g. from mining areas and land used by heavy metal releasing manufacturing industry.

Part B of the research project concerns research on nucleic acids. Modification of the nucleic acid building blocks, especially the nucleobases, is up to now either not very site specific, e.g. complete modification of a certain nucleobase type throughout the whole sequence occurs, or depends on solid-phase synthesis. The latter method is costly for longer nucleic acids and often restricted to the incorporation of modified nucleotides at or near the end of the sequence, again due to economic reasons. The approach described here will allow the site-specific modification of nucleotides in DNA or RNA of unrestricted length. Such specifically modified larger oligonucleotides will then open up new ways to investigate the specific effect of natural modifications and lesions on structure and function of DNA and RNA. In addition, the introduction of specific groups, e.g. primary amines of thiols, can be used to introduce site specific fluorescence labels, e.g. for FRET studies, into long nucleic acid constructs such as ribozymes.

 

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Sequence-specific post-synthetic oligonucleotide labeling for single-molecule fluorescence applications
Egloff David, Oleinich Igor A., Zhao Meng, König Sebastian L. B., Sigel Roland K. O., Freisinger Eva (2016), Sequence-specific post-synthetic oligonucleotide labeling for single-molecule fluorescence applications, in ACS Chem. Biol., 11, 2558-2567.
Investigating the influence of histidine residues on the metal ion binding ability of the wheat metallothionein g-Ec-1 domain
Tarasava Katsiaryna, Freisinger Eva (2015), Investigating the influence of histidine residues on the metal ion binding ability of the wheat metallothionein g-Ec-1 domain, in J. Inorg. Biochem., 153, 197-203.
Sequence-specific generation of 1,N6-ethenoadenine and 3,N4-ethenocytosine in single-stranded unmodified DNA
Egloff David, Oleinich Igor A., Freisinger Eva (2015), Sequence-specific generation of 1,N6-ethenoadenine and 3,N4-ethenocytosine in single-stranded unmodified DNA, in ACS Chem. Biol., 10, 547-553.
An optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins
Tarasava Katsiaryna, Freisinger Eva (2014), An optimized intein-mediated protein ligation approach for the efficient cyclization of cysteine-rich proteins, in Protein Eng. Des. Sel. , 27, 481-488.
Cadmium in metallothioneins
Freisinger Eva, Vašák M. (2013), Cadmium in metallothioneins, in Sigel Astrid, Sigel Helmut (ed.), Springer, Dordrecht, 339-371.
Cu(I) coordination by two plant metallothioneins
Wan X., Schicht O., Freisinger E. (2013), Cu(I) coordination by two plant metallothioneins, in Z. Anorg. Allg. Chem., 639, 1365-1369.
Solution structure of the circular g-domain analog from the wheat metallothionein Ec-1
Tarasava Katsiaryna, Johannsen Silke, Freisinger Eva (2013), Solution structure of the circular g-domain analog from the wheat metallothionein Ec-1, in Molecules, 18, 14414-14429.
Sulfide ions as modulators of metal-thiolate cluster size in a plant metallothionein
Huber T., Freisinger E. (2013), Sulfide ions as modulators of metal-thiolate cluster size in a plant metallothionein, in Dalton Trans., 42, 8878-8889.
Incorporation of sulfide ions into the cadmium(II) thiolate cluster of
Wan X., Freisinger E. (2012), Incorporation of sulfide ions into the cadmium(II) thiolate cluster of, in Inorg. Chem., 52, 785-792.
NMR spectroscopy in bioinorganic chemistry
Donghi D., Johannsen S., Sigel R. K. O., Freisinger E. (2012), NMR spectroscopy in bioinorganic chemistry, in Chimia, 66, 791-797.
Binding of a designed anti-cancer drug to the heart of an RNA three-way junction
Phongtongpasuk S., Paulus S., Schnabl J., Sigel R. K. O., Spingler B., Hannon M. J., Freisinger E., Binding of a designed anti-cancer drug to the heart of an RNA three-way junction, in Angew. Chem. Int. Ed..
Metal ion release from metallothioneins: Proteolysis as an alternative to oxidation
Peroza E. A., Cabral A. C. S, Wan X., Freisinger E., Metal ion release from metallothioneins: Proteolysis as an alternative to oxidation, in Metallomics.
The major function of a metallothionein from the aquatic fungus Heliscus lugdunensis is cadmium detoxification
Loebus J., Leitenmaier B., Meissner D., Braha B., Krauss G.-J., Dobritzsch D., Freisinger E., The major function of a metallothionein from the aquatic fungus Heliscus lugdunensis is cadmium detoxification, in J. Inorg. Biochem..

Collaboration

Group / person Country
Types of collaboration
Prof. Roland K. O. Sigel, University of Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Martin Stillman, University of Western Ontario Canada (North America)
- in-depth/constructive exchanges on approaches, methods or results
Prof. Michael Hannon, University of Birmingham Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Gerd-Joachim Krauß, Dr. Dirk Wesenberg, Martin-Luther-University Halle-Wittenberg Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
Prof. Oliver Zerbe, University of Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Serge Chesnov, Functional Genomics Centre Zurich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Research Infrastructure
Prof. Bernhard Lippert, University of Dortmund Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Jens Müller, Westfälische Wilhelms-University of Münster Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Uwe Karst, Westfälische Wilhelms-University of Münster Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
8th Annual Dorothy Crowfoot-Hodgkin Symposium Poster -natural analogs of a wheat metallothionein 10.10.2013 Zurich, Switzerland Freisinger Eva; Tarasava Katsiaryna;
8th Annual Dorothy Crowfoot-Hodgkin Symposium Poster A modular system for site-specific modifications of native oligonucleotides 10.10.2013 Zurich, Switzerland Oleinich Igor; Freisinger Eva;
COST-CM1105 Whole Action and WG2 Meetin Poster Synthesis of site-specifically functionalized etheno-DNA 09.09.2013 Barcelona, Spain Egloff David; Freisinger Eva;
COST-CM1105 Whole Action and WG2 Meeting Talk given at a conference A modular system for point-specific labeling of native oligonucleotides 09.09.2013 Barcelona, Spain Oleinich Igor; Freisinger Eva;
Swiss Chemical Society Fall Meeting (SCS 2013) Talk given at a conference Non-natural analogs of a wheat metallothionein 06.09.2013 Lausanne, Switzerland Freisinger Eva; Tarasava Katsiaryna;
Swiss Chemical Society Fall Meeting (SCS 2013) Poster Understanding metal ion binding ability, thermodynamic stability and structure of a metallothionein from Musa acuminata (banana) 06.09.2013 Lausanne, Switzerland Jakovleska Jovana; Freisinger Eva;
Swiss Chemical Society Fall Meeting (SCS 2013) Poster A modular system for point-specific labeling of native oligonucleotides 06.09.2013 Lausanne, Switzerland Oleinich Igor; Freisinger Eva;
Swiss Chemical Society Fall Meeting (SCS 2013) Poster Development of a new method for site-specific etheno-DNA synthesis 06.09.2013 Lausanne, Switzerland Freisinger Eva; Egloff David;
XII International Symposium on Inorganic Biochemistry – Collaboration and Beyond Talk given at a conference New metallothionein sequences: Broadening the range of cluster structures 28.08.2013 Wroclaw, Poland Freisinger Eva;
12th International Symposium on Inorganic Biochemistry Poster Understanding the structure of metal complexes formed by a plant metallothionein from Musa acuminata 28.08.2013 Wroclaw, Poland Jakovleska Jovana; Freisinger Eva;
12th International Symposium on Inorganic Biochemistry Talk given at a conference Spectroscopic properties of the fungal metallothionein Neclu_MT2 28.08.2013 Wroclaw, Poland Loebus Jens; Freisinger Eva;
12th International Symposium on Inorganic Biochemistry Talk given at a conference Spectroscopic studies on the CdII specific metallothionein Neclu_MT1 28.08.2013 Wroclaw, Poland Loebus Jens; Leitenmaier Barbara Agnes; Freisinger Eva;
12th International Symposium on Inorganic Biochemistry Poster Artificial analogs of the -domain from the wheat metallothionein Ec-1 28.08.2013 Wroclaw, Poland Freisinger Eva; Tarasava Katsiaryna;
16th International Conference on Biological Inorganic Chemistry (ICBIC 16) Poster Metal ion release from metallothioneins: Competition, oxidation, and proteolysis 22.07.2013 Grenoble, France Freisinger Eva;
16th International Conference on Biological Inorganic Chemistry (ICBIC 16) Talk given at a conference Metal ion release from metallothioneins: Competition, oxidation, and proteolysis 22.07.2013 Grenoble, France Freisinger Eva;
16th International Conference on Biological Inorganic Chemistry (ICBIC 16) Poster Non-natural analogs of a wheat metallothionein 22.07.2013 Grenoble, France Tarasava Katsiaryna; Freisinger Eva;
FEMS 2013 (5th Congress of European Microbiologists) Poster The first CdII specific metallothionein Neclu_MT1 confers CdII tolerance to Heliscus lugdunensis: Physiological and structural characterization 21.07.2013 Germany, Germany Leitenmaier Barbara Agnes; Freisinger Eva; Loebus Jens;
4th International Symposium on Metallomics Talk given at a conference GHOST - A GST hydrogel system for spectroscopic characterization and rapid NMR structure determination of small proteins 08.07.2013 Oviedo, Spain Loebus Jens; Freisinger Eva;
5th European Conference on Chemistry for Life Sciences (5th ECCLS) Poster In-situ formation of fluorescent etheno-nucleobase 10.06.2013 Barcelona, Spain Egloff David; Freisinger Eva;
Symposium on Biomolecular Synthesis and Nanotechnology Poster Modular system for site-specific labeling of native oligonucleotides 03.06.2013 Odene, Denmark Freisinger Eva; Oleinich Igor;
Summer School Chemistry on Metals in Biological Systems Poster Non-natural analogs of a wheat metallothionein 12.05.2013 Louvain-la-Neuve, Belgium Freisinger Eva; Tarasava Katsiaryna;
COST-CM1105 Meeting of Working Group 2 Talk given at a conference Site-specific functionalization of larger nucleic acids 05.03.2013 Birmingham, UK, Great Britain and Northern Ireland Freisinger Eva;
Bioinorganic Chemistry Conference 2013 (Zing Conferences) Talk given at a conference Novel metallothionein forms: Enlarging the range of cluster structures 19.02.2013 Lanzarote, Spain Freisinger Eva;
6th Asian Biological Inorganic Chemistry Conference (AsBIC VI) Talk given at a conference Insights into the clusters of plant metallothioneins 05.11.2012 Hong Kong, China Freisinger Eva;
School of Chemistry & Chemical Engineering, Sun Yat-sen University Individual talk Understanding the structures of plant metallothioneins / Site-specific functionalization of nucleic acids 02.11.2012 Guangzhou, China Freisinger Eva;
7th Annual Dorothy Crowfoot-Hodgkin Symposium Poster Site-directed etheno adenine formation 08.10.2012 Zurich, Switzerland Freisinger Eva; Egloff David;
7th Annual Dorothy Crowfoot-Hodgkin Symposium Poster Cyclic analog of the γ-Ec-1 metallothionein domain 08.10.2012 Zurich, Switzerland Freisinger Eva; Tarasava Katsiaryna;
COST-CM1105 Whole Action Meeting Talk given at a conference Site-specific functionalization of larger nucleic acids 17.09.2012 Granada, Spain Freisinger Eva;
Swiss Chemical Society Fall Meeting (SCS 2012) Talk given at a conference Etheno-adenine generation at specific sites 13.09.2012 Zurich, Switzerland Egloff David; Freisinger Eva;
Swiss Chemical Society Fall Meeting (SCS 2012) Poster A modular system for the post-synthetic point-specific labeling of natural nucleic acids 13.09.2012 Zurich, Switzerland Freisinger Eva; Oleinich Igor;
Swiss Chemical Society Fall Meeting (SCS 2012) Poster Probing of the metallation-process from the ZnII-to the CdII-form of the -Ec-1 metallothionein by NMR spectroscopy 13.09.2012 Zurich, Switzerland Loebus Jens; Freisinger Eva;
Swiss Chemical Society Fall Meeting (SCS 2012) Talk given at a conference Investigations on the metal-sulfide-thiolate cluster of metallothioneins 13.09.2012 Zurich, Switzerland Freisinger Eva; Huber Tamara;
11th European Biological Inorganic Chemistry Conference (EuroBIC 11) Talk given at a conference Understanding the structures of plant metallothioneins 12.09.2012 Granada, Spain Freisinger Eva;
11th European Biological Inorganic Chemistry Conference (EuroBIC 11) Poster Cyclic analog of the γ-Ec-1 metallothionein domain 12.09.2012 Granada, Spain Tarasava Katsiaryna; Freisinger Eva;
11th European Biological Inorganic Chemistry Conference (EuroBIC 11) Talk given at a conference A protein from the aquatic fungus Heliscus lugdunensis is the first cadmium specific metallothionein 12.09.2012 Granada, Spain Loebus Jens; Leitenmaier Barbara Agnes; Freisinger Eva;
40th International Conference on Coordination Chemistry (ICCC 40) Talk given at a conference Spectroscopic insights into the smallest possible metal-thiolate cluster 09.09.2012 Valencia, Spain Freisinger Eva;
International Conference on Magnetic Resonance in Biological Systems Poster GST fusion protein facilitates NMR structure solution and spectrosocpical characterization of small proteins 19.08.2012 Lyon, France Freisinger Eva; Loebus Jens; Leitenmaier Barbara Agnes;
XX International Roundtable on Nucleosides, Nucleotides and Nucleic Acids (XX IRT) Poster A modular system of the incorporation of point-specific modifications into larger nucleic acids 05.08.2012 Quebec, Canada Oleinich Igor; Freisinger Eva;
XX International Roundtable on Nucleosides, Nucleotides and Nucleic Acids (XX IRT) Poster Site-directed etheno adenine formation 05.08.2012 Quebec, Canada Freisinger Eva; Egloff David;
Graduate School, Department of Biology, University of Konstanz Individual talk Metallothioneins: Storage and detoxification of metal ions 12.07.2012 Konstanz, Germany, Germany Freisinger Eva;
International Symposium on Metal Complexes (ISMEC2012) Poster Mechanism of sulfide ion incorporation into the metal cluster of metallothioneins 18.06.2012 Lisbon, Portugal Freisinger Eva; Huber Tamara;


Awards

Title Year
Forschungskredit UZH 2013 2013
Master scholarship of the Werner Foundation of the University of Zurich for Jelena Habjanic 2013
Graduate School CMSZH Travel Award 2012

Associated projects

Number Title Start Funding scheme
105269 Structure investigation of metal clusters in plant metallothioneins with X-ray crystallography and spectroscopic methods 01.11.2004 Project funding (Div. I-III)
119106 A. Structures and properties of plant metallothioneins and related artificial proteins - B. Site specific modifications of larger nucleic acids 01.06.2008 SNSF Professorships
153666 Structures, folding, and metallation pathways of metallothioneins from plants and fungi 01.06.2014 Project funding (Div. I-III)
144964 Upgrade of the UZH NMR Core Facility 01.12.2012 R'EQUIP
119106 A. Structures and properties of plant metallothioneins and related artificial proteins - B. Site specific modifications of larger nucleic acids 01.06.2008 SNSF Professorships
175623 Cluster diversity in plant and fungi metallothioneins - Properties and structures as a gate to functions 01.10.2017 Project funding (Div. I-III)

Abstract

This proposal is divided into two, largely independent projects: Project A focuses on the structures and properties of plant metallothioneins (MTs). A subproject will also investigate cyclic MT forms. The tight regulation of reactive transition metal ions is essential to impede side reactions and cellular damage. One protein superfamily that is taking part in the uptake, transport, and regulation of metal ions within the cell are the MTs, small Cys-rich proteins with the ability to coordinate metal ions with the electron configuration d10 in form of metal clusters. MTs from plants have been scarcely studied so far. In this project we are investigating members of each of the four plant MT subfamilies. We apply mostly spectroscopic techniques such as UV/vis, CD, and atomic absorption spectroscopy coupled with chromatographic techniques for separation and identification purposes, but also more elaborate methods such as EXAFS or PAC spectroscopy are used. Very important for our research is also mass spectrometry. Further, NMR spectroscopy enables us to study metal ion binding affinities or coordination environments of metal ions, and can be used to determine 3D structures. The latter we also try to achieve with crystallographic techniques. These experiments are complemented with functional studies to draw conclusions about the possible roles of plant MTs in vivo. We look at metal ion release under specific conditions, investigate the properties of our proteins in yeast cells as a model system for eukaryotic cells, and even analyse protein expression pattern of a wheat grain specific MT directly in the seed.In project B we are performing site-specific modifications of larger nucleic acids with a DNA-templated approach. We design and synthesize specific reactive groups that are able to generate the desired modification. By coupling these reactive groups to a short complementary oligonucleotide sequence we position them directly opposite the nucleotide to be modified and in this way achieve the desired site-specificity. On the one hand, modifications are introduced that are known mutagenic lesions occurring naturally in DNA such as ?-adducts of bases, oxidized guanine at the C8 position or UV light-induced photo-adducts. Such specifically modified nucleic acids are important for, e.g., biochemical studies of nucleotide repair mechanisms or to monitor the fidelity of polymerases in performing translesional synthesis. In addition we are attempting to introduce functional groups at the N4 position of cytosine bases, which can be used for specific labelling purposes such as the attachment of fluorophores. Our studies are hence important for researchers from the fields of Biochemistry, Structural Biology, Biophysics, and from the Nanosciences alike.
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