Covalent modification of proteins by ubiquitin plays a fundamental role in the control of biological processes and is mediated by the concerted action of E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases. While initially identified as a signal for proteasome-dependent degradation, it is now well established that ubiquitylation can have different impacts on the fate of modified proteins. The diversity of possible outcomes of ubiquitylation seems to be caused by the diversity of possible ubiquitin modifications ranging from mono-ubiquitylation to the attachment of poly-ubiquitin chains where multiple ubiquitin moieties are covalently attached to each other via isopeptide bonds between the C-terminal glycine of one ubiquitin and one out seven lysines of another ubiquitin. Since different ubiquitin chains adopt different topologies, an obvious possibility is that proteins exist that selectively interact with different ubiquitin chains and that these proteins determine the specific fate of the modified proteins. To identify such proteins, we will employ unnatural amino acids to synthesize defined ubiquitin chains by a combination of recombinant protein expression and chemical approaches. The respective chains will be used as affinity matrix to isolate proteins, which recognize ubiquitin chains in a linkage-specific manner. In addition, modification with ubiquitin may alter the ability of proteins to interact with other proteins. To address this possibility, we will use the tumor suppressor p53 as model substrate, since p53 is known to be mono- and poly-ubiquitylated as well as modified by the ubiquitin-like proteins SUMO and NEDD8. Respective lysine residues of p53 and the C-terminal glycine of ubiquitin, SUMO, and NEDD8 will be replaced by unnatural amino acids that can be linked by “click-chemistry”. The binding properties of the respectively modified p53 molecules will then be analyzed in coprecipitation experiments. Finally, unnatural amino acids will be used to address the hypothesis that the "ubiquitin loading" status of E2 and/or E3s affects their interaction. To this end, the active site cysteine residue of a given E2 (e.g. UbcH5B) or E3 (E6AP) will be replaced by unnatural amino acids that can be linked to ubiquitin by click-chemistry. The conjugates will be used to study the interaction of E2s and E3s by various means including NMR and/or X-ray crystallography, if feasible. In conclusion, the proposed studies will contribute to the elucidation of the mechanisms involved in determining the fate of ubiquitylated proteins and in controlling E2-E3 interaction.