Expanding the genetic code – protein chemistry in living systems

Orateur : Kathrin LANG (Technical University of Munich, Germany)

The desire to study and manipulate biological processes in their native environment has fuelled the development of approaches to endow proteins with new chemical functionalities in vivo. Genetic code expansion allows the site-specific incorporation of artificial amino acids into virtually any protein in living cells and animals. Together with significant developments in designing and re-discovering chemical reactions that are amenable to physiological conditions and applicable in living systems these methods have begun to have a direct impact on studying biological processes. In this talk I will discuss how proteins can be site-specifically equipped with unnatural amino acids (UUAs) bearing electrophilic moieties that are inert under physiological conditions but covalently link positions that are brought into proximity as a result of protein-protein interactions and protein complex formation. Such proximity-triggered crosslinking approaches (chemical crosslinking) allow covalent stabilization of low-affinity and transient protein complexes in living bacteria and mammalian cells and we have pioneered their use to aid structural elucidation of previously inaccessible transient, low-affinity protein complexes. Furthermore I will present a novel approach that utilizes site-specifically incorporated UAAs as a platform for chemoenzymatic reactions. Combining genetic code expansion, bioorthogonal chemistries and a transpeptidation reaction we have developed a novel and generally applicable tool to ubiquitylate target proteins in an inducible fashion. Our approach allows the site-specific attachment of ubiquitin and ubiquitin-like modifiers - via a native isopeptide bond – to non-refoldable, multi-domain proteins under native conditions. This method enables for the first time the site-specific, inducible and E1/E2/ E3-independent ubiquitylation of proteins in living mammalian cells, providing a powerful tool to dissect the biological functions of ubiquitylation with temporal control.

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