Semisynthesis of ubiquitylated proteins.

Most, if not all, proteins are at one point or another posttranslationally modified so as to regulate their biological function. One of the most common protein modifications is ubiquitylation, in which the small protein ubiquitin is attached to a target protein in a multistep process involving dedicated ubiquitin ligases. Ubiquitylation is best known for its role in protein turnover. In this case, attachment of a polyubiquitin chain to a target protein leads to its eventual destruction by the 26S proteasome. However, attachment of ubiquitin is not always a kiss of death for the recipient protein; it is increasingly clear that the modification plays additional roles, including regulating protein trafficking and protein signaling. Understanding these functions at the molecular level necessitates that we have access to homogenous ubiquitylated proteins, something that has proved very difficult using standard biochemical approaches. In this chapter, we describe the development of synthetic chemistries and protein semisynthesis methods that permit the site-specific ubiquitylation of proteins. The utility of this methodology is illustrated through the synthesis of ubiquitylated histones.