Small-molecules are ideal tools for manipulating and observing protein function in living cells, but developing specific and potent probes is a major challenge. Alternative strategies to knock down protein function exist but such experiments can run into roadblocks if even a small proportion of the protein is still active. In contrast, in a gain-of-function study, even very low enzyme activation efficiency is usually enough to turn on a signaling pathway. This may yield more mechanistic insights, explains Peng Chen of Peking University in China.

One type of gain-of-function approach involves the use of photocleavable 'caging' groups to turn off functional amino acids in proteins. When hit with light, the caging group is stripped away, restoring native protein activity. A handful of photocleavable unnatural amino acids are available that can be site-specifically incorporated into proteins in living cells using genetic code expansion methods. But the ultraviolet light required to trigger 'decaging' can cause surface-receptor internalization, altered intracellular signaling and cytotoxicity.

Chen and his colleagues now provide an alternative chemical decaging strategy, one that adds to the protein function manipulation toolbox and may provide advantages over light-based decaging in many situations. “The toughest challenge we met was to find the proper bioorthogonal protection group and deprotection reagents (the caging and decaging pair), which can be used on intact proteins inside living cells,” says Chen. “Although many deprotection methods have been developed for amino acids and peptides, most of them have to be carried out under harsh reaction conditions that are not suitable for biological applications.” After extensively combing the chemical literature for an appropriate reaction, the researchers settled on a palladium catalyst–mediated depropargylation reaction.

Chemical decaging of an unnatural amino acid using a palladium catalyst enables restoration of native protein function. Figure from Li et al., Nature Publishing Group.

But first, the team needed to show that decaging was robust. Initially, they optimized the decaging reaction in vitro, showing that it was efficient and did not cause any damage to the protein GFP. They then moved the reaction in vivo, decaging a fluorescent small molecule and GFP in mammalian cells, demonstrating that the palladium catalysts could enter living cells and that the decaging reaction was not toxic and was reasonably efficient.

Chen's team then truly put the method to the test by focusing on OspF, a phosphothreonine lyase that is secreted into host cells by pathogenic Shigella microbes. Once in the host cell, OspF irreversibly dephosphorylates mitogen-activated protein kinases such as phosphorylated Erk. The team introduced propargyl-lysine at position 134 in OspF's active site and expressed the protein in mammalian cells. Following decaging, they observed a restoration of OspF's dephosphorylation function. They tagged OspF and Erk in turn with GFP to follow their intracellular localization; OspF remained in the nucleus after decaging, but dephosphorylated Erk did not, suggesting a possible mechanism for OspF's virulence in host cells.

Though it remains to be tested, the chemical decaging strategy is likely to be general and may find use in tissues and possibly even animals. Chen points out that tools for genetically incorporating other unnatural propargyl-caged residues into proteins are already available. “This, in conjunction with the general applicability of the palladium-mediated deprotection chemistry, should allow our chemical decaging strategy to be expanded to other amino acids,” he says.