Genetically Encoded Optochemical Probes for Simultaneous Fluorescence Reporting and Light Activation of Protein Function with Two-Photon Excitation

Ji Luo,Rajendra Uprety,Yuta Naro,Alexander Deiters,Chungjung Chou,Jason W Chin,Duy P Nguyen

doi:10.1021/ja5055862

Abstract

The site-specific incorporation of three new coumarin lysine analogues into proteins was achieved in bacterial and mammalian cells using an engineered pyrrolysyl-tRNA synthetase system. The genetically encoded coumarin lysines were successfully applied as fluorescent cellular probes for protein localization and for the optical activation of protein function. As a proof-of-principle, photoregulation of firefly luciferase was achieved in live cells by caging a key lysine residue, and excellent OFF to ON light-switching ratios were observed. Furthermore, two-photon and single-photon optochemical control of EGFP maturation was demonstrated, enabling the use of different, potentially orthogonal excitation wavelengths (365, 405, and 760 nm) for the sequential activation of protein function in live cells. These results demonstrate that coumarin lysines are a new and valuable class of optical probes that can be used for the investigation and regulation of protein structure, dynamics, function, and localization in live cells. The small size of coumarin, the site-specific incorporation, the application as both a light-activated caging group and as a fluorescent probe, and the broad range of excitation wavelengths are advantageous over other genetically encoded photocontrol systems and provide a precise and multifunctional tool for cellular biology.

Highlights

Good photochemical properties, chemical stability, and ease of synthesis make coumarins an important class of fluorescent probes for biological studies.[1−3] In addition to being versatile fluorophores, coumarin chromophores can be used as lightremovable protecting groups, so-called “caging groups”, that are photolyzed through one- and two-photon irradiation.[4]
We report the site-specific incorporation of three coumarin amino acids into proteins via genetic code expansion with unnatural amino acids (UAAs)[14−16] to integrate the optical properties of coumarin probes into cellular systems
The active site of the PylRS can be further engineered through directed evolution to enable the incorporation of additional unnatural amino acids with new functions, including posttranslational modifications, bioconjugation handles, photocross-linkers, photocaging groups, and others.[14]

Summary

■ INTRODUCTION

Chemical stability, and ease of synthesis make coumarins an important class of fluorescent probes for biological studies.[1−3] In addition to being versatile fluorophores, coumarin chromophores can be used as lightremovable protecting groups, so-called “caging groups”, that are photolyzed through one- and two-photon irradiation.[4]. We report the site-specific incorporation of three coumarin amino acids into proteins via genetic code expansion with unnatural amino acids (UAAs)[14−16] to integrate the optical properties of coumarin probes into cellular systems. The photochemical characteristics of these UAAs complement and enhance the properties of caged and fluorescent amino acids that have been genetically encoded in bacterial and mammalian cells.[19−25] Lysines 1−3 were assembled in three steps from their corresponding coumarin alcohols (Supporting Information, Scheme S1). Coumarin lysines 1 and 2 can be used as both fluorescent and lightactivated probes for optochemical control of protein function using UV or near-IR light, while coumarin lysine 3 may serve as a stable fluorescent probe that does not decage under UV excitation

■ RESULTS AND DISCUSSION

■ SUMMARY

■ ACKNOWLEDGMENTS

■ REFERENCES