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dc.contributor.advisor Neumann, Heinz Prof. Dr.
dc.contributor.author Hahn, Liljan
dc.date.accessioned 2015-10-28T09:33:41Z
dc.date.available 2015-10-28T09:33:41Z
dc.date.issued 2015-10-28
dc.identifier.uri http://hdl.handle.net/11858/00-1735-0000-0023-9661-9
dc.language.iso eng de
dc.relation.uri http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject.ddc 572 de
dc.title Investigation of Nucleosome Dynamics by Genetic Code Expansion de
dc.type doctoralThesis de
dc.contributor.referee Neumann, Heinz Prof. Dr.
dc.date.examination 2015-03-10
dc.description.abstracteng The DNA of eukarytic cells is compacted into a complex macromolecular structure called chromatin. Histones are the small structural proteins that form central octamers around which the DNA is wrapped to form the basic repeating units of chromatin, the nucleosomes. The histone proteins are highly altered by a plethora of posttranslational modifications (PTMs) which are involved in altering the chromatin structure affecting various cellular mechanisms. Acetylation of lysine residues neutralizes the positive charge thereby disturbing contacts with DNA or other histones. Recently, other acyl modifications, including lysine propionylation, crotonylation or butyrylation have been discovered. In this thesis the site-specific incorporation of those novel acyl modifications was addressed using genetic code expansion. This technique is used to incorporate unnatural amino acids (UAAs) in response to an amber stop codon. The codon is recognized by a pair of evolved aminoacyl-tRNA synthetase/tRNACUA that are orthogonal components for the recognition of specific UAAs. Mutant synthetases were evolved to recognize acyl lysine derivatives as a substrate and were selected from a MbPylS library. The histone H4 tail was reported to be most extensively modified by the different acyl modifications. Since H4 was hitherto only unsuccessfully addressed by genetic code expansion, a new strategy which overcame these limitations is presented here. Furthermore the yield of site-specifically modified H4 was efficient enough to allow for the reconstitution of octamers and nucleosomal arrays carrying the modified H4. Thereby, we provide a basis to study effects caused by these acyl modifications. We could identify deacylation activity for the E. coli HDAC CobB, indicating different reaction velocities depending upon the type of acyl modification. In further explorations using genetic code expansion technologies, we set out to explore the highly dynamic nucleosome structure by monitoring time resolved structural changes by fluorescence resonance energy transfer (FRET). However, this approach requires the installation of two distinct fluorophores, which remains challenging. In this thesis, the site-specific installation of FRET pairs on histones is addressed based on a combinatorial approach combining the labeling of an UAA carrying a functional group suitable for derivatization by click chemistry with the thiol labeling of an individually placed cysteine. de
dc.contributor.coReferee Diederichsen, Ulf Prof. Dr.
dc.contributor.thirdReferee Ficner, Ralf Prof. Dr.
dc.subject.eng Genetic code expansion de
dc.subject.eng Unnatural amino acids de
dc.subject.eng PTM (posttranslational modifications) de
dc.subject.eng Lysine acylations de
dc.subject.eng Propionylation de
dc.subject.eng Crotonylation de
dc.subject.eng Butyrylation de
dc.subject.eng Acetylation de
dc.subject.eng Click chemistry de
dc.subject.eng Protein labeling de
dc.subject.eng FRET de
dc.identifier.urn urn:nbn:de:gbv:7-11858/00-1735-0000-0023-9661-9-2
dc.affiliation.institute Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) de
dc.subject.gokfull Biologie (PPN619462639) de
dc.identifier.ppn 837983339

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