The ubiquitin�proteasome system serves a crucial role in maintaining appropriate protein levels in eukaryotic cells through proteolysis. The vast majority of proteasomal substrates are ubiquitinated to some degree. However, the extent to which the ubiquitination patterns are able to promote proteasomal degradation of a substrate is still unclear. The ubiquitin signal itself is complex, as ubiquitin chains may link through any of seven positions on the ubiquitin molecule, and form a multitude of complex structures.Herein, we describe the development of a method to produce polyubiquitin chains with a high degree of control using a combination of expressed protein ligation, non-natural amino acid incorporation and the Staudinger ligation. The Staudinger ligation is a chemoselective reaction that occurs between a phosphine and an azide that ultimately produces an amide bond. This traceless reaction produces an authentic isopeptide bond between an azide-labeled proximal ubiquitin and a distal ubiquitin labeled with a C-terminal phosphinothioester. The distal and proximal ubiquitins are prepared using a combination of chemical and biological techniques to yield unique functionalities that allow for a chemoselective Staudinger ligation that joins the two monomers. Importantly, the ligations can be performed iteratively to produce any ubiquitin chain with a high degree of control. To date, we have used this method to produce dimers linked through Lys48 and Lys63. This method has the potential to produce ubiquitin chains of any architecture, allowing for an unrestricted study of the ubiquitin signal.

Convergent semisynthetic route to authentic ubiquitin chains

248th National Meeting (2014)

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