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Structures, folding, and metallation pathways of metallothioneins from plants and fungi

Applicant Freisinger Eva
Number 153666
Funding scheme Project funding (Div. I-III)
Research institution Institut für Chemie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Inorganic Chemistry
Start/End 01.06.2014 - 30.09.2017
Approved amount 400'000.00
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All Disciplines (5)

Discipline
Inorganic Chemistry
Physical Chemistry
Biophysics
Molecular Biology
Biochemistry

Keywords (8)

Protein folding; Spectroscopy; Cyclic proteins; Fluorescence; NMR; Metallothioneins; Plants; Fungi

Lay Summary (German)

Lead
Metallothionine (MTs) sind kleine Metallionen-bindende Proteine, welche in nahezu allen Lebewesen gefunden werden. Im Gegensatz zu den MTs der Säugetiere und Vertebraten wurden die entsprechenden Formen der Pflanzen und Pilze bisher nur spärlich untersucht. Dies überrascht, da diese MTs einen aussergewöhnlichen Reichtum an unterschiedlichen Aminosäuresequenzen aufweisen, welcher bisher noch nicht mit Strukturen und Eigenschaften korreliert werden konnte. Das Schliessen dieser Lücke ist das zentrale Thema des vorliegenden Projektes, welches Methoden der Bioanorganischen Chemie mit solchen aus der Strukturbiologie vereinigt.
Lay summary

Dieses Projekt befasst sich mit der Thematik der Metallothionine (MTs) aus der Sicht der Bioanorganischen Chemie. MTs weisen einen hohen Cystein (Cys) Gehalt auf. Die Thiolatgruppen dieser Cys-Reste sind äusserst effiziente Liganden für sogenannte weiche Metallionen. Zu diesen gehören die für die meisten Lebewesen essentiellen Zn(II) und Cu(I) Ionen aber auch die in der Regel toxischen Cd(II) und Hg(II) Ionen. Aufgrund dieser ausgeprägten Metallbindungseigenschaften wird angenommen, dass MTs eine Rolle in der Metallionenhomeostase und –entgiftung spielen.

In diesem Projekt sollen die Metallbindungseigenschaften spezifischer MTs der Pflanzen und Pilze untersucht werden. Die pflanzlichen MTs sind besonders interessant, da verschiedene, von der Aminosäuresequenz her recht unterschiedliche MT-Formen innerhalb der gleichen Pflanze vorkommen und es noch keine Hinweise dafür gibt, weshalb die Pflanze solch verschiedene MTs benötigt. Das Pilz-MT wurde aus einem Süsswasser-Hyphomyzeten isoliert, welcher in einer Schwermetall-belasteten Quelle wächst und dessen Produktion spezifisch durch Cd(II) Ionen angeregt werden kann. Die Sequenz-spezifischen Hintergründe dieser Spezifität, wenn vorhanden, sollen untersucht werden. Wir werden bevorzugt Techniken der optischen Spektroskopie anwenden, da diese sich hervorragend zur Untersuchung der Metallbindung eignen, sowie ferner NMR, Fluoreszenz- und Röntgenabsorptionsspektroskopie.

Die Ergebnisse dieses Projektes werden uns dabei helfen zu verstehen, wie Organismen dazu in der Lage sind, Spezifität in der Selektion und Bindung der verschiedenen Metallionen zu erreichen. Diese vertiefte Einsicht in die Metallhomeostase und Entgiftungsvorgänge kann zum Beispiel eine Grundlage dafür sein, Nahrungspflanzen zu entwickeln, welche einen besonders hohen Anteil an essentiellen Metallionen, wie z.B. Zn(II) Ionen, aufweisen, oder auch spezielle Pflanzen oder Pilze zur Entgiftung von Böden und Wasser, die sogenannte Phytoremediation, zu verwenden.
Direct link to Lay Summary Last update: 06.05.2014

Responsible applicant and co-applicants

Employees

Publications

Publication
Localization and spectroscopic analysis of the Cu(I) binding site in the wheat metallothionein Ec-1
Tarasava Katsiaryna, Loebus Jens, Freisinger Eva (2016), Localization and spectroscopic analysis of the Cu(I) binding site in the wheat metallothionein Ec-1, in Int. J. Mol. Sci. , 17, 371.
Oxidation of the N-terminal domain of the wheat metallothionein Ec-1 leads to the formation of three distinct disulfide bridges
Tarasava Katsiaryna, Chesnov Serge, Freisinger Eva (2016), Oxidation of the N-terminal domain of the wheat metallothionein Ec-1 leads to the formation of three distinct disulfide bridges, in Biopolymers: Peptide Sci. , 106, 295-308.
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.
Further insights into the metal ion binding abilities and the metalation pathway of a plant metallothionein from Musa acuminata
Cabral Augusto, Jakovleska Jovana, Deb A., Penner-Hahn J., Pecoraro Vincent, Freisinger Eva, Further insights into the metal ion binding abilities and the metalation pathway of a plant metallothionein from Musa acuminata, in J. Biol. Inorg. Che..

Collaboration

Group / person Country
Types of collaboration
Prof. Frank Neese, Max-Planck-Institut für chem. Energiekonversion, Mülheim Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Serge Chesnov, Functional Genomics Centre Zurich Switzerland (Europe)
- Publication
- Research Infrastructure
Prof. Oliver Zerbe, University of Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Gerd-Joachim Krauß, Dr. Dirk Dobritzsch, Martin-Luther-University Halle-Wittenberg Germany (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Dr. Matteo Tegoni Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Lars Hemmingsen, University of Copenhagen Denmark (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Martin Stillman Canada (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Swiss Chemical Society Fall Meeting (SCS 2017) Poster Potentiometry as a tool to study metal ion coordination to metallothioneins 21.08.2017 Bern, Switzerland Freisinger Eva; Perinelli Monica;
Swiss Chemical Society Fall Meeting (SCS 2017) Poster Plant metallothionein 2 protein: Following the metalation process 21.08.2017 Bern, Switzerland Freisinger Eva; Salim Alma;
18th International Conference on Biological Inorganic Chemistry (ICBIC 18) Talk given at a conference Metallothioneins recruiting additional players: The influence of histidine 31.07.2017 Florianopolis, Brazil Freisinger Eva;
14th International Symposium on Applied Bioinorganic Chemistry (ISABC 14) Talk given at a conference "on-off" – Metal ion binding and release in metallothioneins 07.06.2017 Toulouse, France Freisinger Eva;
14th International Symposium on Applied Bioinorganic Chemistry (ISABC 14) Poster Metalation pathway of a plant metallothionein 07.06.2017 Toulouse, France Freisinger Eva; Perinelli Monica;
14th International Symposium on Applied Bioinorganic Chemistry (ISABC 14) Poster Metalation pathway of a plant metallothionein 07.06.2017 Toulouse, France Salim Alma; Freisinger Eva;
6th Georgian Bay International Conference on Bioinorganic Chemistry (CanBIC 6) Talk given at a conference Methods to tackle the metal ion coordination sites in metallothioneins 23.05.2017 Parry Sound, Ontario, Canada Freisinger Eva;
FEBS Practical and Lecture Course “Chemistry of Metals in Biological Systems Poster Plant metallothionein 2 protein: Following the metalation process 21.05.2017 Louvain-la-Neuve, Belgium Freisinger Eva; Salim Alma;
8th Asian Biological Inorganic Chemistry Conference (AsBIC 8) Talk given at a conference Spectroscopic and structural studies of histidine-rich metallothioneins 04.12.2016 Auckland, New Zealand Freisinger Eva;
11th Annual Dorothy Crowfoot-Hodgkin Symposium Poster Further insights into the cadmium specifity of Neclu_MT 03.10.2016 Zurich, Switzerland Perinelli Monica; Freisinger Eva;
11th Annual Dorothy Crowfoot-Hodgkin Symposium Poster 'Step by step' structural investigation of a plant metallothioneins 2 protein 03.10.2016 Zurich, Switzerland Freisinger Eva; Salim Alma;
Swiss Chemical Society Fall Meeting (SCS 2016) Poster Further insights into the cadmium specifity of Neclu_MT 15.09.2016 Zurich, Switzerland Perinelli Monica; Freisinger Eva;
13th European Biological Inorganic Chemistry Conference (EuroBIC 13) Poster 'Step by step' structural investigation of a plant metallothioneins 2 protein 28.08.2016 Budapest, Hungary Freisinger Eva; Salim Alma;
13th European Biological Inorganic Chemistry Conference (EuroBIC 13) Talk given at a conference Beyond mammalian metallothioneins 28.08.2016 Budapest, Hungary Freisinger Eva;
10th Annual Dorothy Crowfoot-Hodgkin Symposium Poster Tackling the structure of the metal binding domains of a plant metallothionein 3 05.10.2015 Zurich, Switzerland Freisinger Eva; Jakovleska Jovana;
10th Annual Dorothy Crowfoot-Hodgkin Symposium Poster Investigating the structure of the metallothionein 2 protein from the plant Cicer arietinum 05.10.2015 Zurich, Switzerland Salim Alma; Freisinger Eva;
; Swiss Chemical Society Fall Meeting (SCS 2015) Poster Investigating the structure of the metallothionein 2 protein from the plant Cicer arietinum 04.09.2015 Lausanne, Switzerland Freisinger Eva; Salim Alma;
Swiss Chemical Society Fall Meeting (SCS 2015) Poster Tackling the structure of the metal binding domains of a plant metallothionein 3 04.09.2015 Lausanne, Switzerland Freisinger Eva; Jakovleska Jovana;
XIII International Symposium on Inorganic Biochemistry Talk given at a conference Like the HK-variant, the structure and metal ion binding abilities of the g-domain of the wheat Ec-1 metallothionein remain unperturbed in view of a multitude of manipulations 01.09.2015 Karpacz, Poland Freisinger Eva;
17th International Conference on Biological Inorganic Chemistry (ICBIC 17) Talk given at a conference New metallothionein forms: Metal ion coordination beyond the enigmatic metal-thiolate clusters 20.07.2015 Beijing, China Freisinger Eva;
13th International Symposium on Applied Bioinorganic Chemistry (ISABC 13) Talk given at a conference Using a peptidic M(II)2S6 cluster to study and manipulate metal ion binding abilities 20.06.2015 Galway, Ireland Freisinger Eva;
9th Annual Dorothy Crowfoot-Hodgkin Symposium Poster Biologically relevant or an artifact? The copper binding site in wheat metallothionein 06.10.2014 Zurich, Switzerland Freisinger Eva;
Swiss Chemical Society Fall Meeting (SCS 2014) Poster Coordination of metal ions by the two cysteine-rich domains of the plant metallothionein-3 from Musa acuminata (banana) 11.09.2014 Zurich, Switzerland Jakovleska Jovana; Freisinger Eva;
Swiss Chemical Society Fall Meeting (SCS 2014) Poster Biologically relevant or an artifact? The copper binding site in wheat metallothionein 11.09.2014 Zurich, Switzerland Freisinger Eva;
12th European Biological Inorganic Chemistry Conference (EuroBIC 12) Poster Towards the structure-function characterization of a metal binding plant metallothionein from Musa acuminata (banana) 24.08.2014 Zurich, Switzerland Freisinger Eva; Jakovleska Jovana;
12th European Biological Inorganic Chemistry Conference (EuroBIC 12) Poster Biologically relevant or an artifact? The copper binding site in wheat metallothionein 24.08.2014 Zurich, Switzerland Freisinger Eva;
12th European Biological Inorganic Chemistry Conference (EuroBIC 12) Poster Investigating a dominant Zn2Cd species of the fungal metallothionein Neclu_MT2 24.08.2014 Zurich, Switzerland Freisinger Eva;
Institute of Inorganic Chemistry (Prof. U. Simon) Individual talk Plant and fungal metallothioneins viewed from a bioinorganic perspective / A modular system for sequence-specific nucleic acid modifications 02.07.2014 Aachen, Germany Freisinger Eva;


Self-organised

Title Date Place
12th European Biological Inorganic Chemistry Conference (EuroBIC 12) 24.08.2014 Zurich, Switzerland

Awards

Title Year
SBIC Travel Grant (€ 200): 14th International Symposium on Applied Bioinorganic Chemistry (ISABC 14), Toulouse, France, June 7-10, 2017 2017
SBIC Travel Grant (€ 255): FEBS Practical and Lecture Course “Chemistry of Metals in Biological Systems”, Louvain-la-Neuve, Belgium, May 21-28, 2017 2017

Associated projects

Number Title Start Funding scheme
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
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)
105269 Structure investigation of metal clusters in plant metallothioneins with X-ray crystallography and spectroscopic methods 01.11.2004 Project funding (Div. I-III)

Abstract

The proposed project is placed within the field of metallothioneins (MTs) and in particular concentrates on the plant forms of MTs as well as one fungal MT. Part of the project was already included in my SNSF-Professorship grant (06/2008-05/2014, PP00P2_139194), but could not or will not be finalized mostly due to personnel reasons until the end of my SNSF-Professorship. The parts are indicated in the text at the respective positions, and often lay the ground work for the other studies proposed here.The proposed research plan is divided into four AIMS, each dealing with a particular MT form, but all aims have in common that a special emphasis is placed on the structures and folding mechanisms of the proteins to advance our knowledge towards a structure-function relationship.AIM I focuses on the determination of the structure, folding, and metallation pathway of chickpea MT2, an MT with a single metal-thiolate cluster and the typical long linker region found in plant MTs from the MT1, MT2, and MT3 sub-families. NMR spectroscopy, fluorescence studies, and a combination of proteolytic digestion studies with analytical protein and metal ion quantification methods will be applied to address this goal. AIM II will investigate the separate Cys-rich regions of banana MT3. Here the method of potentiometric pH titrations newly established in our group will help to identify coordination sites and to calculate stability constants. NMR spectroscopy will be again applied to analyze the three-dimensional structures of the respective metal-peptide complexes. AIM III is a continuation of our recently started work on neclu_MT1, an MT from the aquatic fungus Heliscus lugdunensis which shows CdII-specificity. Again we will use NMR spectroscopy to evaluate the metal ion binding sites as well as the structure of the protein, but we will also apply potentiometric pH titrations to identify coordinating ligands during the metallation process of this rather small MT form. AIM IV finally again is focused on plant MTs, specifically on the Ec subfamily. We indent to analyze the folding of wheat Ec-1, the structure of which was previously determined in our group, and will apply both intrinsic fluorescence of Trp residues that were point-specifically introduced into the amino acid sequence as well as the measurement of FRET efficiencies of a fusion protein with N- and C-terminal expressed fluorescence tags to observe structural changes upon metallation. A second part of AIM IV will focus on the structure determination of the two Ec proteins from Arabidopsis thaliana, which where shown to have different properties towards ZnII and CdII in yeast complementation assays. These differences most likely originate from differences in metal ion coordination.Our research is settled within the field of bioinorganic chemistry combining aspects of coordination chemistry with structural biology. We use a multitude of spectroscopic as well as other analytical and biochemical methods for our studies. Results from this project will have considerable impact for the field of plant MT research and the bioinorganic community in general.
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