Investigation of Nucleosome Dynamics by Genetic Code Expansion

Hahn, Liljan

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.identifier.uri	http://dx.doi.org/10.53846/goediss-5337
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-5337
dc.language.iso	eng	de
dc.rights.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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GGNB - Göttinger Graduiertenzentrum für Neurowissenschaften, Biophysik und molekulare Biowissenschaften [1114]
GGNB - Göttingen Graduate Center for Neurosciences, Biophysics and Molecular Biosciences

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