Genetic Encoding of a Photocaged Glutamate for Optical Control of Protein Functions

Genetic encoding of photocaged noncanonical amino acids provides a powerful tool to study protein functions through optical control but is not yet available for acidic amino acids. Herein, we report the first site-specific genetic encoding of a photocaged glutamate, 4-methoxy-7-nitroindolinyl caged glutamate (MNI-Glu), into recombinant proteins via an expanded genetic code through evolved EcLeuRS/tRNA pair. Using two enzymes as examples, we demonstrate that substituting the conserved-active-site glutamate of a secreted alkaline phosphatase and a protease HRV3C to MNI-Glu allows photoregulatory control of their enzymatic activities. Our approach is an important addition to the photocaged noncanonical amino-acid toolbox and provides a general method to photocontrol protein activity based on caging a critical glutamate. [GRAPHICS] .

Automated summary

We report the first site-specific genetic encoding of a photocaged glutamate, 4-methoxy-7-nitroindolinyl caged glutamate (MNI-Glu), into recombinant proteins via an expanded genetic code. By substituting the conserved-active-site glutamate of two enzymes with MNI-Glu, we demonstrate photoregulatory control of their enzymatic activities. Our approach is a valuable addition to the photocaged noncanonical amino-acid toolbox and provides a general method for photocontrolling protein activity based on caging a critical glutamate.