Project

Discipline

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

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.

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Last update: 21.02.2013

Active participation

Title	Year

Number	Title	Start	Funding scheme

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.