Targeted poly(ADP-ribosyl)ation of proteins (Gezielte Poly(ADP-Ribosyl)ierung von Proteinen)

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ADP-ribosylation and poly(ADP-ribosy)lation are reversible posttranslational modifications of proteins catalyzed by ADP-ribosyltransferases (ARTs) like poly(ADP-ribose) polymerases (PARPs) using NAD+ as substrate. Poly(ADP-ribose) (PAR) is a linear or multibranched polyanion of variable size that can interact noncovalently with numerous proteins. PAR formation has been associated with DNA repair, maintenance of genomic stability, transcription, telomere regulation, cell division, energy metabolism, cell death, tumour suppression and ageing. However, our knowledge of how PAR and ADP-ribose modifications modulate the properties and regulate the function of their target proteins is sparse. For a better understanding of these modifications and their biological role we propose the synthesis of proteins with distinct sites of mono- and poly(ADP-ribosy)lation. As first target we chose histone protein H1.2, which is mono- and poly(ADP-ribosy)lated in vivo and involved in the compaction of chromatin and regulation of gene expression. The generated functionalized proteins will be of major interest and the basis for comprehensive biochemical follow-up studies. We will reach the project goal by incorporation of a novel unnatural amino acids that bears a protected alkoxyamine functionality via stop codon suppression. This will enable site-specific modification of proteins with orthogonally reacting functional groups after liberation of the alkoxyamine functionality. Subsequently, new ligation chemistry will be developed to offer a powerful tool to conjugate the unnatural amino acid site-specifically with the reducing end of ADP-ribose and PAR. Such well-defined functionalized proteins will be used to investigate the biological role of mono- and poly(ADP-ribosy)lation of histone H1.2, whose PAR-modification has been suggested as a regulator of chromatin structure.