Limits...
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.

Show MeSH

Related in: MedlinePlus

Engineering of an optochemicallycontrolled Photinus pyralis firefly luciferase throughunnatural amino acid mutagenesis. (A)Caging groups at position K206 are blocking access to the bindingpocket by luciferin and ATP and are disrupting a required hydrogenbonding network. (B) After decaging, wild-type Fluc is generated andthe substrates can now enter the active site. PDB: 2D1S. (C) Bright-Gloluciferase assay of cells that were either kept in the dark or irradiated(365 nm, 4 min). Chemiluminescence units were normalized to the −UAA/–UVcontrol. No enzymatic activity was observed for the caged proteins,and significant increases in luminescence were observed after photolysisof luciferase containing 1 or 2, while theK206 → 3 mutant was permanently deactivated, asexpected. Error bars represent standard deviations from three independentexperiments.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4333581&req=5

fig3: Engineering of an optochemicallycontrolled Photinus pyralis firefly luciferase throughunnatural amino acid mutagenesis. (A)Caging groups at position K206 are blocking access to the bindingpocket by luciferin and ATP and are disrupting a required hydrogenbonding network. (B) After decaging, wild-type Fluc is generated andthe substrates can now enter the active site. PDB: 2D1S. (C) Bright-Gloluciferase assay of cells that were either kept in the dark or irradiated(365 nm, 4 min). Chemiluminescence units were normalized to the −UAA/–UVcontrol. No enzymatic activity was observed for the caged proteins,and significant increases in luminescence were observed after photolysisof luciferase containing 1 or 2, while theK206 → 3 mutant was permanently deactivated, asexpected. Error bars represent standard deviations from three independentexperiments.

Mentions: To apply the coumarin lysines 1–3 in the optical control of protein function in live cells,fireflyluciferase (Fluc) was selected as an initial target because bioluminescencemeasurements afford low background, high sensitivity, and easy quantification.On the basis of the Fluc crystal structure, a critical lysine residue,K206, was identified, which is positioned at the edge of the substrate-bindingpocket (Figure 3B). It has been proposed thatthis residue stabilizes and orients ATP in the active site.45,46 The ε-amino group on K206 provides a hydrogen-bond interactionwith the γ-phosphate of ATP and promotes the adenylation reactionwith luciferin, thus being essential for catalytic activity as shownby the dramatic decrease in enzymatic activity displayed by the K206Rmutant.45 Therefore, we hypothesized thata sterically demanding coumarin caging group placed on K206 wouldprevent the interaction with ATP and limit the overall access of thesubstrates to the active site (Figure 3A).Photolysis of the coumarin lysine would remove the caging group andproduce a native lysine residue, restoring the catalytic activityof the enzyme (Figure 3B). A genetically encodedphotocaged lysine at K206 would enable the enhanced regulation ofthe catalytic activity of firefly luciferase via light activation.


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)

Engineering of an optochemicallycontrolled Photinus pyralis firefly luciferase throughunnatural amino acid mutagenesis. (A)Caging groups at position K206 are blocking access to the bindingpocket by luciferin and ATP and are disrupting a required hydrogenbonding network. (B) After decaging, wild-type Fluc is generated andthe substrates can now enter the active site. PDB: 2D1S. (C) Bright-Gloluciferase assay of cells that were either kept in the dark or irradiated(365 nm, 4 min). Chemiluminescence units were normalized to the −UAA/–UVcontrol. No enzymatic activity was observed for the caged proteins,and significant increases in luminescence were observed after photolysisof luciferase containing 1 or 2, while theK206 → 3 mutant was permanently deactivated, asexpected. Error bars represent standard deviations from three independentexperiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4333581&req=5

fig3: Engineering of an optochemicallycontrolled Photinus pyralis firefly luciferase throughunnatural amino acid mutagenesis. (A)Caging groups at position K206 are blocking access to the bindingpocket by luciferin and ATP and are disrupting a required hydrogenbonding network. (B) After decaging, wild-type Fluc is generated andthe substrates can now enter the active site. PDB: 2D1S. (C) Bright-Gloluciferase assay of cells that were either kept in the dark or irradiated(365 nm, 4 min). Chemiluminescence units were normalized to the −UAA/–UVcontrol. No enzymatic activity was observed for the caged proteins,and significant increases in luminescence were observed after photolysisof luciferase containing 1 or 2, while theK206 → 3 mutant was permanently deactivated, asexpected. Error bars represent standard deviations from three independentexperiments.
Mentions: To apply the coumarin lysines 1–3 in the optical control of protein function in live cells,fireflyluciferase (Fluc) was selected as an initial target because bioluminescencemeasurements afford low background, high sensitivity, and easy quantification.On the basis of the Fluc crystal structure, a critical lysine residue,K206, was identified, which is positioned at the edge of the substrate-bindingpocket (Figure 3B). It has been proposed thatthis residue stabilizes and orients ATP in the active site.45,46 The ε-amino group on K206 provides a hydrogen-bond interactionwith the γ-phosphate of ATP and promotes the adenylation reactionwith luciferin, thus being essential for catalytic activity as shownby the dramatic decrease in enzymatic activity displayed by the K206Rmutant.45 Therefore, we hypothesized thata sterically demanding coumarin caging group placed on K206 wouldprevent the interaction with ATP and limit the overall access of thesubstrates to the active site (Figure 3A).Photolysis of the coumarin lysine would remove the caging group andproduce a native lysine residue, restoring the catalytic activityof the enzyme (Figure 3B). A genetically encodedphotocaged lysine at K206 would enable the enhanced regulation ofthe catalytic activity of firefly luciferase via light activation.

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