Metal containing peptides as specific DNA binders
by Harita Rao
Date of Examination:2015-11-09
Date of issue:2016-01-29
Advisor:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Franc Meyer
Referee:Prof. Dr. Sofi Elmroth
Persistent Address:
http://dx.doi.org/10.53846/goediss-5490
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Abstract
English
This doctoral thesis involves design and synthesis of small model peptides based on appended domains of DNA binding proteins that are found to be crucial in generating a particular cellular response or function. The model peptides include metal binding sites due to their well-defined geometry and ability to stabilize the entire protein structure around the coordination sphere of the metal ion. Moreover, peptidomimetic scaffolds based on chimeric metal complex peptide conjugates offer a possibility to harness the recognition properties of natural metalloenzymes and hence, assist in performing biochemical reactions in a sequence specific manner. In this direction two main projects were pursued during the doctoral thesis. In the first project, platinum complex/peptide chimera mimicking the integration host factor (IHF) were synthesized and tested for their DNA binding abilities. IHF is a DNA architectural protein which sequence specifically binds to DNA and induces a bend > 160˚ in dsDNA through minor grove intercalation. The motivation behind the synthesis of such chimerical peptides is to enhance site specific targeting of cisplatin by means of peptide-DNA recognition. Enhanced binding to DNA sequences containing a GG site was observed with the IHF mimicking Pt complex/peptide chimera consisting of the cyclic minor grove recognizing unit, the positively charged lysine dendrimer and the platinum chelating unit contained within the glycine linker tethering the two peptidic units. Though it was not possible to attain sequence specific recognition with the platinated IHF mimics, these provide good lead structures to begin with. Further optimization of the design offers the possibility to generate a library of Pt complex/peptide chimera as potential chemotherapeutics. The second project involves design of artificial zinc finger nucleases for site specific DNA cleavage. Due to their modular design and high affinity towards cognate DNA the zinc finger domain proteins can be applied for engineering artificial nucleases by incorporation of additional metal binding sites within its structure. The artificial amino acid residues can be functionalized to introduce multiple metal binding sites within the protein domains. In this study, the Zif268 sequence was modified to contain a histidine mimicking amino acid bearing a triazacyclononane (TACN) ligand. The TACN ligand provides an allosteric Zn(II) coordination site and is expected to assist cleavage of the DNA phosphodiester backbone. The Zif268 domain consisting of a tandem repeat of three zinc fingers was prepared by following a semi-synthetic strategy that utilizes intein mediated protein ligation. Initial DNA cleavage experiments suggest insufficient DNA cleavage ability of the modified zinc finger constructs. Further optimization of the design of the modified Zif268 domain is necessary in order to improve their nuclease activity.
Keywords: platinum complex; peptide chimera; zinc fingers
