# Recent Advances in Protein Caging Tools for Protein Photoactivation
## 3 Site-Specific Protein Caging  
As a method for site-specific introduction of the photodegradable protecting group, the use of the cysteine residue of the proteins has been utilized [11; 30; 31; 32; 33; 34]. The selective reactivity of the thiol group realized the site-specific caging of the cysteine residue of which there are often one or a few on protein surfaces. Similar to the random amine coupling, the function of intact proteins was reported to be photo-regulated by caging with thiol-reactive protecting reagents when the cysteine residues were at the sites involved in protein functions [30; 31]. By this approach, the enzymatic activities of myosin [30] and \(\beta\)-galactosidase [31] were demonstrated to be activated by light exposure. As another method using the thiol-based reaction, the caging of thiophosphorylated proteins was also reported [32]. In this method, the threonine residue of a protein was thiophosphorylated using 3-phosphoinositide-dependent kinase, and then modified with a thiol-reactive caging reagent. After photolysis, the caged protein converted to the thiophosphorylated protein which exerted almost the same activity as the phosphorylated one. This kinase-coupled approach may be versatile for caging a variety of phosphorylated proteins. To apply such thiol-based site-specific caging approaches to any other proteins, the use of cysteine-substituted mutants has been widely utilized (Figure 2A) [11; 33; 34]. In this approach, the function-related site is replaced with cysteine, and a caging reagent that selectively reacts  
Figure 1: Schematic illustration of caging of compounds and proteins. (**A**) Caging of small molecular compounds. (**B**) Successful and (**C**) unsuccessful caging of proteins through random modification of lysine residues. with the thiol group is applied. Based on this approach, the actin polymerization activity of cofilin, which was unknown so far, was exerted by light exposure in living cells, giving new insights into the role of cofilin [11]. Furthermore, light-induced depolymerization of actin was also achieved by caged cofilin on the glass substrate [33], and the pore-formation on cellular membranes was photo-induced by caged \(\alpha\)-hemolysin [34]. Although this approach is simple to introduce a protecting group at the desired active site, the proteins produced after photolysis are only the cysteine-substituted mutant protein on which the mutation is introduced at a critical site involved in function (Figure 2A). Therefore, there is a high risk that the light-induced activity of the mutant protein will be inferior to that of the native one. Thus, the caging strategy based on post-translational chemical modification often requires the site-specific replacement with reactive amino acids for selective caging, and therefore, almost exclusively applied to mutant proteins. As a method for site-specific protein caging, another genetic approach using unnatural caged amino acids has been widely employed [5, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53]. Using the genetic code extension technology such as notably amber mutation and four-base codon mutation [43], in which an unnatural amino acid is coded to a specific codon, the desired position of the protein of interest can be replaced with a caged amino acid (Figure 2B). Until now, caged aspartic acid [35, 39], caged serine [36, 51], caged glycine [37, 43], caged tyrosine [5, 38, 40, 48, 50], caged cysteine [42, 47], caged lysine [44, 49, 52, 53], caged phosphoserine [46] and caged  
Figure 2: Schematic illustration of site-specific caging of proteins. (**A**) Caging based on cysteine mutagenesis. (**B**) Caging based on unnatural amino acid introduction using genetic code extension technologies. (**C**) Caging based on protein semi-synthesis using expressed protein ligation (EPL). (**D**) Caging based on ligand-directed chemistry.