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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 119106
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 01.06.2008 - 31.05.2012
Approved amount 1'527'628.00
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All Disciplines (4)

Discipline
Inorganic Chemistry
Molecular Biology
Biophysics
Biochemistry

Keywords (12)

Plant metallothioneins; Artificial proteins; Metalloselenoneins; Cyclic proteins; Site specific nucleobase modifications; DNA-templated synthesis; Spectroscopy; X-ray crystallography; metal-thiolate clusters; bioinorganic chemistry; nucleic acids; site-specific modifications

Lay Summary (English)

Lead
Lay summary
This scientific project is divided into two, largely independent subjects. focuses on the metal ion binding abilities of plant metallothioneins (MTs) and related artificial proteins, including the investigation of their structures and general properties. Metal ions are essential for life but depending on the concentration and sort of metal ion they can also exhibit a considerable amount of toxicity. The tight regulation of reactive transition metal ions resulting in free metal ion concentrations of below one ion per cell is essential for the survival of any living organism. One protein superfamily that is taking part in these uptake, transport and accumulation mechanisms inside the cell are the MTs. Generally, MTs are a family of small proteins with an outstandingly high content of the amino acid cysteine and metal ions with the electronic configuration d10. In a way, MTs might be regarded as inorganic metal-thiolate clusters embedded in a biological matrix and are thus one of the classical examples out of the research field of Bioinorganic Chemistry. MTs from plants have been scarcely studied so far. They have a proposed role in metal ion homeostasis, detoxification, and plant development, although final evidence in this regard is still pending. Differing considerably in their primary amino acid sequences from the well-studied mammalian isoforms, different properties and new exciting three-dimensional structures can be foreseen. This new knowledge might even 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. 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.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Protein and metal cluster structure of the wheat metallothionein domain -Ec-1: the second part of the puzzle
Loebus Jens, Peroza Estevao A., Blüthgen Nancy, Fow Thomas, Meyer-Klaucke Wolfram, Zerbe Oliver, Freisinger Eva (2011), Protein and metal cluster structure of the wheat metallothionein domain -Ec-1: the second part of the puzzle, in J. Biol. Inorg. Chem., 16, 683-694.
Structural features specific to plant metallothioneins
Freisinger Eva (2011), Structural features specific to plant metallothioneins, in J. Biol. Inorg. Chem., 16(7), 1035-1045.
The metal-thiolate clusters of plant metallothioneins
Freisinger Eva (2010), The metal-thiolate clusters of plant metallothioneins, in Chimia, 64, 217-224.
Metallothioneins in plants
Freisinger Eva (2009), Metallothioneins in plants, in Met. Ions Life Sci., 5, 107-153.
The plant metallothionein 2 from Cicer arietinum forms a single metal–thiolate cluster
Wan Xiaoqiong, Freisinger Eva (2009), The plant metallothionein 2 from Cicer arietinum forms a single metal–thiolate cluster, in Metallomics, 1, 489-500.
The β(E)-domain of wheat Ec-1 metallothionein: A metal-binding domain with a distinctive structure
Peroza Estevao A., Schmucki Roland, Güntert Peter, Freisinger Eva, Zerbe Oliver (2009), The β(E)-domain of wheat Ec-1 metallothionein: A metal-binding domain with a distinctive structure, in J. Mol. Biol., 387, 207-218.
Plant MTs—long neglected members of the metallothionein superfamily
Freisinger Eva (2008), Plant MTs—long neglected members of the metallothionein superfamily, in Dalton Trans., 6663-6675.
Spectroscopic characterization of Cicer arietinum metallothionein 1
Schicht Oliver, Freisinger Eva (2008), Spectroscopic characterization of Cicer arietinum metallothionein 1, in Inorg. Chim Acta, 362, 714-724.
The two distinctive metal ion binding domains of the wheat metallothionein Ec-1
Peroza Estevao A., Al Kaabi Ali, Meyer-Klaucke Wolfram, Wellenreuther Gerd, Freisinger Eva (2008), The two distinctive metal ion binding domains of the wheat metallothionein Ec-1, in J. Inorg. Biochem., 103, 342-353.
Cadmium in metallothioneins
Freisinger Eva, Vasak Milan, Cadmium in metallothioneins, in Met. Ions Life Sci., 11.

Collaboration

Group / person Country
Types of collaboration
Martin-Luther-Universität Halle-Wittenberg Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel
University of Western Ontario United States of America (North America)
- Publication
University of Copenhagen Denmark (Europe)
- Publication
University of Konstanz Germany (Europe)
- Publication
Westfälische Wilhelms-Universität Münster Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
- Exchange of personnel

Awards

Title Year
Bester Vortrag GdCh Doktorandenseminar 2011 in Hohenroda (Analytische Chemie)
CMSZH Travel Award 2012
Forschungskredit 2011

Associated projects

Number Title Start Funding scheme
175623 Cluster diversity in plant and fungi metallothioneins - Properties and structures as a gate to functions 01.10.2017 Project funding (Div. I-III)
139194 A. Structures and properties of plant metallothioneins and related artificial proteins - B. Site specific modifications of larger nucleic acids 01.06.2012 SNSF Professorships
153666 Structures, folding, and metallation pathways of metallothioneins from plants and fungi 01.06.2014 Project funding (Div. I-III)
105269 Structure investigation of metal clusters in plant metallothioneins with X-ray crystallography and spectroscopic methods 01.11.2004 Project funding (Div. I-III)
113728 Structures and properties of plant metallothioneins 01.11.2006 Project funding (Div. I-III)
139194 A. Structures and properties of plant metallothioneins and related artificial proteins - B. Site specific modifications of larger nucleic acids 01.06.2012 SNSF Professorships

Abstract

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. Part of the work on plant MTs is already the subject of the Habilitation I am currently pursuing and was/is funded by grants of the Swiss National Science Foundation (200021-105269/1, 2004-2006; 200020-113728/1, 2006-2008) as well as of the Forschungskredit of the University of Zurich (2004-2006). Plant MTs are a fascinating class of proteins, as on the one hand they have a specific but mostly still unknown role in metal ion homeostasis, detoxification, and plant development, and on the other hand are ideal targets to investigate the formation and coordination chemistry of metal-thiolate and -sulfide clusters. Special emphasis will be put on the determination of the first three-dimensional structures of plant MTs of the four different subfamilies with the aid of X-ray crystallography or NMR spectroscopy. Further techniques applied for the investigation of the proteins include a variety of spectroscopic methods, such as UV-Vis, circular dicroism, NMR and ESR spectroscopy to name just a few. Additionally, mass spectrometry is applied to monitor protein purity and integrity and to investigate cluster formation and metal ion exchange processes. A number of biochemical techniques complement the plethora of methods used. This work will be continued and driven forward over the next years. With respect to the artificial proteins, we will on the one hand study the properties of cyclic MT analogs, a project we recently initiated. To the best of our knowledge, the investigation of cyclic MTs has so far never been undertaken. On the other hand, the preparation of metalloselenoneins (MSs) will be attempted. Up to now, only the synthesis of one CuI-MS form has been described, dating back 16 years. For these two novel MT-related artificial proteins, interesting alterations of structure and properties are expected, which promise to give deeper insights into MT structure and coordination chemistry as well as to open up routes to possible applications in Structural Biology and Pharmaceutical Sciences. The project described in Part B builds on my doctoral and postdoctoral work. 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 make use of DNA-templates to incorporate modifications site-specifically into an oligonucleotide of unrestricted length. The basic technique for this approach is already reasonably established, but so far solely applied for cleavage and cross-linking experiments. The aim of the project described here is to use this method to insert special modifications such as, e.g., (i) DNA lesions for the study of repair and lesion tolerance mechanisms, (ii) post-translational occurring RNA modifications to enable the investigation of RNA structure and function in its native state, and (iii) generally modified artificial building blocks with specific properties such as special metal ion binding abilities or which enable their use as sensors. 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.As outlined on the following pages, our research centers within the field of Bioinorganic Chemistry reaching from the Coordination Chemistry of metal-thiolate clusters to the synthetic chemical modification of nucleotides. From this basis, our work extends into the fields of Structural Biology, Biophysics, Toxicology, Pharmacological Sciences and even Nanoscience.
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