Formation of ubiquitin dimers via azide-alkyne click reaction.

The conjugation of poly-ubiquitin chains is a widespread post-translational modification of proteins that plays a role in many different cellular processes. Notably, the biological function of the attached ubiquitin chain depends on which lysine residue is used for chain formation. Here, we report a method for the modular synthesis of site-specifically linked ubiquitin dimers, which is based on click reaction between two artificial amino acids. In this way, it is possible to synthesize all seven naturally occurring ubiquitin connectivities, thus giving access to all ubiquitin dimers. Furthermore, this method can be generally applied to link ubiquitin to any substrate protein or even to link any two proteins site specifically.