Constructing Photoactivatable Protein with Genetically Encoded Photocaged Glutamic Acid

Abstract

Genetically replacing an essential residue with the corresponding photocaged analogues via genetic code expansion (GCE) constitutes a useful and unique strategy to directly and effectively generate photoactivatable proteins. However, the application of this strategy is severely hampered by the limited number of encoded photocaged proteinogenic amino acids. Herein, we report the genetic incorporation of photocaged glutamic acid analogues in E. coli and mammalian cells and demonstrate their use in constructing photoactivatable variants of various fluorescent proteins and SpyCatcher. We believe genetically encoded photocaged Glu would significantly promote the design and application of photoactivatable proteins in many areas.

Noncanonical Amino Acids in Biocatalysis

“…Genetic code expansion has been used to install protected analogues of functional amino acids into enzymes. These caged amino acids offer an effective strategy to inhibit enzyme activity, either through the steric hindrance of ligand binding sites or by masking key catalytic groups, with facile restoration of activity achievable through cleavage of the obstructing group using chemical − or photochemical − methods.…”

“…Alternatively, GCE provides a more targeted approach to install new regulatory elements, and has been used to incorporate photocaged analogues of Tyr, Lys, Ser and Cys into enzyme active sites. − An early example from the Schultz lab reported the replacement of an active site Tyr503 with O -NBTyr in β-galactosidase to afford an inhibited variant that could be activated upon irradiation with 365 nm light to recover 67% of WT activity . This approach was extended to the development of orthogonal translation components for incorporating photocaged tyrosine analogues in eukaryotic hosts, which has enabled photochemical control of enzymes such Cre recombinases, proteases, or nucleases .…”