CHAPTER 5.2: Genetic Code Expansion Approaches to Introduce Artificial Covalent Bonds into Proteins In Vivo

The desire to study and manipulate biological processes in their native environment has fueled the development of approaches to endow proteins with new chemical groups in vivo. Genetic code expansion allows the site-specific incorporation of artificial, designer amino acids into virtually any protein in living cells and animals. Together with significant developments in designing and rediscovering chemistries that are amenable to physiological conditions and applicable in living systems (in vivo chemistries), these methods have begun to have a direct impact on studies of biological processes that are difficult or impossible to address by more traditional approaches. An emerging area with enormous potential is the site-specific incorporation of unnatural amino acids with functional groups that allow subsequent covalent modification of the protein with biophysical probes and small molecules via chemoselective reactions. This provides an important approach for imaging protein location in live cells and can also be used for controlling an enzyme's activity in vivo. Likewise, the co-translational site-specific incorporation of unnatural amino acids bearing new functional groups that are inert under physiological conditions, but become reactive to form covalent linkages with biomolecules in their vicinity upon a certain trigger, has become an important approach for understanding protein–protein interactions in live cells. This chapter discusses how genetic code expansion approaches together with in vivo chemistries can be utilized to introduce artificial covalent linkages into proteins and how these methods can be used for studying and controlling protein localization and activity and also for mapping and characterizing protein–protein interactions.