Fast phosphine-activated control of protein function using unnatural lysine analogues

Effective, general methods for conditionally activating proteins in their native biological environments are highly useful for biological studies. Since phosphines and azides are not found in pro- and eukaryotic cells, the Staudinger reduction can function as an excellent small molecule-controlled switch for protein activation. This methodology involves site-specifically incorporating azidobenyl-lysine analogues into proteins in live cells. When placed at a crucial position, these unnatural side chains block protein function until a phosphine trigger is added. We discuss methods for expressing caged proteins in bacterial and mammalian cells in high yields, and activating the proteins with an optimized phosphine trigger. We also discuss important considerations for safe and effective synthesis of these molecules. This methodology was used to translocate proteins to the nucleus and to turn-on a protein post-translational modification (SUMOylation) in living cells.