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Genetically encoded optochemical probes for simultaneous fluorescence reporting and light activation of protein function with two-photon excitation.

Luo J, Uprety R, Naro Y, Chou C, Nguyen DP, Chin JW, Deiters A - J. Am. Chem. Soc. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.

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.

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(A) Structures of the genetically encodedcoumarin amino acidsfor fluorescence reporting and light activation of protein function.(B) Crystal structure of PylRS (2Q7H) with the pyrrolysine substrate (yellow)in the active site. (C) Structure of BhcKRS with 1 (green)docked into the active site. Dashed blue lines represent H-bond interactions.(D) SDS-PAGE analysis of sfGFP-Y151TAG containing 1–3 through incorporation in E. coli. The gelwas stained with Coomassie blue (top), and coumarin fluorescence wasimaged via excitation at 365 nm (bottom). (E) Fluorescence micrographsof HEK 293T cells expressing the BhcKRS/tRNACUA pair andmCherry-TAG-EGFP-HA in the presence or absence of 1–3. (F) Western blot analysis of cell lysates using an anti-HAantibody and a GAPDH antibody as a loading control. Full-length proteinexpression is only observed in the presence of 1–3, and incorporation efficiency with all three amino acidsis similar in mammalian cells.
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fig1: (A) Structures of the genetically encodedcoumarin amino acidsfor fluorescence reporting and light activation of protein function.(B) Crystal structure of PylRS (2Q7H) with the pyrrolysine substrate (yellow)in the active site. (C) Structure of BhcKRS with 1 (green)docked into the active site. Dashed blue lines represent H-bond interactions.(D) SDS-PAGE analysis of sfGFP-Y151TAG containing 1–3 through incorporation in E. coli. The gelwas stained with Coomassie blue (top), and coumarin fluorescence wasimaged via excitation at 365 nm (bottom). (E) Fluorescence micrographsof HEK 293T cells expressing the BhcKRS/tRNACUA pair andmCherry-TAG-EGFP-HA in the presence or absence of 1–3. (F) Western blot analysis of cell lysates using an anti-HAantibody and a GAPDH antibody as a loading control. Full-length proteinexpression is only observed in the presence of 1–3, and incorporation efficiency with all three amino acidsis similar in mammalian cells.

Mentions: Here we report the site-specific incorporation of three coumarinamino acids into proteins via genetic code expansion with unnaturalamino acids (UAAs)14−16 to integrate the optical properties of coumarin probesinto cellular systems. Genetic code expansion requires the additionof orthogonal translational machinery to achieve site-specific UAAincorporation into proteins. Recent advances in engineering pyrrolysyl-tRNAsynthetase/tRNA pairs for the incorporation of sterically demandingamino acids17−20 prompted us to synthesize coumarin lysines 1–3 (Figure 1A) and to test their incorporationinto proteins. The photochemical characteristics of these UAAs complementand enhance the properties of caged and fluorescent amino acids thathave been genetically encoded in bacterial and mammalian cells.19−25 Lysines 1–3 were assembled in threesteps from their corresponding coumarin alcohols (Supporting Information, Scheme S1). Briefly, the coumarinalcohols were activated with nitrophenyl chloroformate and coupledto commercially available Boc-lysine. A global deprotection underacidic conditions furnished the corresponding coumarin derivatives 1–3 in good yields.


Genetically encoded optochemical probes for simultaneous fluorescence reporting and light activation of protein function with two-photon excitation.

Luo J, Uprety R, Naro Y, Chou C, Nguyen DP, Chin JW, Deiters A - J. Am. Chem. Soc. (2014)

(A) Structures of the genetically encodedcoumarin amino acidsfor fluorescence reporting and light activation of protein function.(B) Crystal structure of PylRS (2Q7H) with the pyrrolysine substrate (yellow)in the active site. (C) Structure of BhcKRS with 1 (green)docked into the active site. Dashed blue lines represent H-bond interactions.(D) SDS-PAGE analysis of sfGFP-Y151TAG containing 1–3 through incorporation in E. coli. The gelwas stained with Coomassie blue (top), and coumarin fluorescence wasimaged via excitation at 365 nm (bottom). (E) Fluorescence micrographsof HEK 293T cells expressing the BhcKRS/tRNACUA pair andmCherry-TAG-EGFP-HA in the presence or absence of 1–3. (F) Western blot analysis of cell lysates using an anti-HAantibody and a GAPDH antibody as a loading control. Full-length proteinexpression is only observed in the presence of 1–3, and incorporation efficiency with all three amino acidsis similar in mammalian cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4333581&req=5

fig1: (A) Structures of the genetically encodedcoumarin amino acidsfor fluorescence reporting and light activation of protein function.(B) Crystal structure of PylRS (2Q7H) with the pyrrolysine substrate (yellow)in the active site. (C) Structure of BhcKRS with 1 (green)docked into the active site. Dashed blue lines represent H-bond interactions.(D) SDS-PAGE analysis of sfGFP-Y151TAG containing 1–3 through incorporation in E. coli. The gelwas stained with Coomassie blue (top), and coumarin fluorescence wasimaged via excitation at 365 nm (bottom). (E) Fluorescence micrographsof HEK 293T cells expressing the BhcKRS/tRNACUA pair andmCherry-TAG-EGFP-HA in the presence or absence of 1–3. (F) Western blot analysis of cell lysates using an anti-HAantibody and a GAPDH antibody as a loading control. Full-length proteinexpression is only observed in the presence of 1–3, and incorporation efficiency with all three amino acidsis similar in mammalian cells.
Mentions: Here we report the site-specific incorporation of three coumarinamino acids into proteins via genetic code expansion with unnaturalamino acids (UAAs)14−16 to integrate the optical properties of coumarin probesinto cellular systems. Genetic code expansion requires the additionof orthogonal translational machinery to achieve site-specific UAAincorporation into proteins. Recent advances in engineering pyrrolysyl-tRNAsynthetase/tRNA pairs for the incorporation of sterically demandingamino acids17−20 prompted us to synthesize coumarin lysines 1–3 (Figure 1A) and to test their incorporationinto proteins. The photochemical characteristics of these UAAs complementand enhance the properties of caged and fluorescent amino acids thathave been genetically encoded in bacterial and mammalian cells.19−25 Lysines 1–3 were assembled in threesteps from their corresponding coumarin alcohols (Supporting Information, Scheme S1). Briefly, the coumarinalcohols were activated with nitrophenyl chloroformate and coupledto commercially available Boc-lysine. A global deprotection underacidic conditions furnished the corresponding coumarin derivatives 1–3 in good yields.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.

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.

Show MeSH
Related in: MedlinePlus