Publikationsserver der RWTH Aachen University
Neue Anwendungen von (S)-Adenosyl-L-methionin-Analoga mit Protein-Methyltransferasen und Click-Chemie zur sequenzspezifischen Markierung von Proteinen
Abstract
dc:descriptionPosttranslational protein methylation has been connected to important regulatory mechanisms in cells. Especially in context with the histone code, the methylation of arginin and lysine residues on histone proteins and their function in the regulation of gene expression has been studied in detail over the past years. The ubiquitous cofactor S-adenosyl-L-methionin (AdoMet) is the major methyl donor for enzymatic methylation reactions. Protein methyltransferases (MTases) catalyse methyl group transfer to target amino acid residues. However, the methyl group is a poor chemical reporter. To transfer larger functional groups than the methyl group to target positions, synthetic AdoMet analogues have been synthesized which have already been used with DNA-MTases for sequence-specific modification of DNA. The AdoMet analogues carry extended, activated side chains replacing the methyl group. With these synthetic cofactor analogues and protein MTases, a two-step labeling system for proteins was established in this work. Target proteins shall be modified by transferring the extended side chains carrying functional groups from synthetic AdoMet analogues with protein MTases. The functional groups are subsequently labeled in a bioorthogonal chemical reaction. The fungal histone lysine protein MTase Dim-5 is specific for H3K9 and was chosen for test reactions with the synthetic AdoMet analogues. A fluorescein-labeled histone peptide encompassing amino acids 1-15 from histone H3 served as a substrate. Extended side chains from synthetic AdoMet analogues could be transferred to the peptide by Dim-5. A new cofactor with a terminal alkyne as the functional group was synthesized particularly for the application in the chemical labeling reaction. The extended side chain of this AdoMet analogue AdoEnIn was transferred to the histone peptide by Dim-5. By transferring the extended side chain, a terminal alkyne was enzymatically introduced to the substrate peptide. An azide-derivatized reporter can be coupled with the alkinylated peptide in a Cu(I) catalyzed cycloaddition reaction, a so called ‘Click-Reaction’. This coupling reaction is of bioorthogonal character and can be applied to biological systems in vitro. Using the click reaction, the alkinylated peptide was biotinylated. After optimizing the reaction conditions for the enzymatic side chain transfer and the subsequent chemical biotinylation with the histone peptide, the modification-and-labeling system should be applied to full length proteins. Histone H3 was modified with the AdoEnIn side chain by Dim-5 and biotinylated via the click reaction. The reaction mixture was seperated by SDS-PAGE and detected by a Western-Blot using peroxidase-coupled avidin. The analogue AdoEnIn could also serve as a cofactor for eukaryotic protein MTases. In contrast to prokaryotic protein MTases, these are only active in protein complexes with several subunits. A human ASH2 complex contains MLL4, a H3K4 protein MTase. Histone H3 was alkinylated with AdoEnIn by the ASH2 complex and biotinylated via the click reaction. The biotinylated histone was detected by a western-blot using the peroxidase-avidin conjugate. A C-terminal fragment of MLL4 carrying the catalytic site was able to modify histone H3 with the AdoEnIn side chain, chemical biotinylation could be detected in a western-blot. The enzymatic activities of endogenous protein MTases in cell extracts were used for AdoEnIn side chain transfer to their specific substrates. Labeling with biotin allowed the detection of the modified proteins in western blots. In addition, the biotinylated proteins could either be isolated with streptavidin-coated magnetic beads and subsequently silver stained in SDS gels or were alternatively analyzed by mass spectrometry.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2009
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Peters, Wibke
- Contributors dc:contributor
-
- Weinhold, Elmar
Subjects
dc:subject × 9Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger