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

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.

Small-molecule control of protein function through Staudinger reduction

“…Since individual rate constants of several of these steps are unknown, we decided to compare the overall kinetics to the optical activation of an EGFP-K85 mutant containing a caged coumarin-lysine (Supplementary Figure S8). Here, in agreement with previously observed EGFP maturation kinetics, 37,38 a t ½ of 35 min was observed, indicating that, as expected, the fluorescent protein activation with the small molecule trigger is slightly slower than the light-activation. Thus, genetically encoded OABK in conjunction with small molecule activation allows for the conditional regulation of intracellular protein maturation.…”

“…37 The lysine K85 in EGFP was targeted for OABK introduction, since it undergoes crucial electrostatic and H-bonding interactions with a nearby aspartate, serine, and cysteine, all key residues for chromophore maturation and fluorescence (Figure 2a). HEK293T cells were co-transfected with pEGPF-K85TAG-mCherry and pOABKRS-4PylT in the presence of OABK .…”

Playing with the Molecules of Life

“…246–249 These photocaged ncAAs have been used to control kinase, protease, intein, and other enzyme activities, nuclear localization, virus-host interactions, and cell signaling cascades. 102, 163, 240, 250–255 …”

A photoactivatable Src homology 2 (SH2) domain

“…Among all the PylRS variants examined (Fig. S1, ESI †), DizPKRS, 12 BhcKRS, 13 and TcokRS, 14 showed excellent efficiency in the incorporation of ONBK into GFP UV -Asn149 mutant protein in response to amber nonsense codon (Fig. 2B).…”