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Modulating protein activity using tethered ligands with mutually exclusive binding sites.

Schena A, Griss R, Johnsson K - Nat Commun (2015)

Bottom Line: We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector.The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector.Tethering such a ligand to the protein of interest results in an intramolecular ligand-protein interaction that can be disrupted through the presence of the effector.

View Article: PubMed Central - PubMed

Affiliation: 1] École Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering, Avenue Forel 2, EPFL SB ISIC LIP BCH-4303, CH-1015 Lausanne, Switzerland [2] École Polytechnique Fédérale de Lausanne, Institute of Bioengineering, CH-1015 Lausanne, Switzerland [3] National Centre of Competence in Research in Chemical Biology, CH-1015 Lausanne, Switzerland.

ABSTRACT
The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand-protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors.

No MeSH data available.


Related in: MedlinePlus

Control of HCA by streptavidin.(a) Crystal structure of the active site of HCA bound to a benzenesulfonamide (PDB ID 1CNW). (b) Modulation of HCA activity in CLASH-Strep/HCA. A fusion protein of SNAP-tag, a 30-proline linker, NanoLuc Luciferase (NLuc) and HCA and is labelled with a synthetic molecule containing a fluorophore (red star) and the two intramolecular ligands benzenesulfonamide (SA) and biotin (B). Binding of streptavidin (Strep) to the tethered B displaces SA from HCA, leading both to a conformational change that can be seen by a decrease in BRET efficiency, and to an increase of enzymatic activity. (c) Chemical structure of the labelling compound. (d) Enzymatic activity of HCA and followed by measuring absorbance at 348 nm. Streptavidin (Strep) acts as an effector of the hydrolytic activity of HCA: in the absence of Strep, SA binds to and inhibits HCA (black dashed line), while on addition of saturation concentrations of Strep, the catalytic activity increases (black solid line). (e) Emission spectra of 10 nM CLASH-Strep/HCA at increasing streptavidin concentrations. (f) Titration of 10 nM CLASH-Strep/HCA with streptavidin. As a control, the synthetic ligand BG-Cy3-SA lacking the tethered biotin is used.
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f2: Control of HCA by streptavidin.(a) Crystal structure of the active site of HCA bound to a benzenesulfonamide (PDB ID 1CNW). (b) Modulation of HCA activity in CLASH-Strep/HCA. A fusion protein of SNAP-tag, a 30-proline linker, NanoLuc Luciferase (NLuc) and HCA and is labelled with a synthetic molecule containing a fluorophore (red star) and the two intramolecular ligands benzenesulfonamide (SA) and biotin (B). Binding of streptavidin (Strep) to the tethered B displaces SA from HCA, leading both to a conformational change that can be seen by a decrease in BRET efficiency, and to an increase of enzymatic activity. (c) Chemical structure of the labelling compound. (d) Enzymatic activity of HCA and followed by measuring absorbance at 348 nm. Streptavidin (Strep) acts as an effector of the hydrolytic activity of HCA: in the absence of Strep, SA binds to and inhibits HCA (black dashed line), while on addition of saturation concentrations of Strep, the catalytic activity increases (black solid line). (e) Emission spectra of 10 nM CLASH-Strep/HCA at increasing streptavidin concentrations. (f) Titration of 10 nM CLASH-Strep/HCA with streptavidin. As a control, the synthetic ligand BG-Cy3-SA lacking the tethered biotin is used.

Mentions: To demonstrate the generality of CLASH, we applied it to regulate the activity of the enzyme human carbonic anhydrase II (HCA). Furthermore, we wanted to translate the activity modulation into a signal compatible with biosensing. We synthesized a dual ligand in which the HCA inhibitor benzenesulfonamide and a biotin moiety are connected in a way that binding to their respective protein receptors would be mutually exclusive (Fig. 2a–c) and used again streptavidin as the effector. The choice of the linker composition and length in the labelling molecule was based on the crystal structure of the receptor proteins. To couple HCA activity switching to a readout compatible with biosensing, we expressed HCA as a fusion protein with the luciferase NanoLuc, a 30-proline linker (PP30) and SNAP-tag, following the design principles of LUCIDs—our previously described class of BRET-based biosensors18. The SNAP-PP30-NLuc-HCA fusion protein was labelled with the synthetic ligand also containing the fluorophore Cy3 for the BRET readout. The resulting semisynthetic protein CLASH-Strep/HCA (Fig. 2b) is expected to show an increase in HCA activity and display a change in BRET efficiency between NanoLuc and Cy3 when the tethered ligand is displaced from HCA through binding of the effector streptavidin. The interaction between streptavidin and biotin has a Kd in the femtomolar range; since this is much lower than the concentration of CLASH-Strept/HCA in the assay, the transition of the titration curve occurs around the concentration of the CLASH construct.


Modulating protein activity using tethered ligands with mutually exclusive binding sites.

Schena A, Griss R, Johnsson K - Nat Commun (2015)

Control of HCA by streptavidin.(a) Crystal structure of the active site of HCA bound to a benzenesulfonamide (PDB ID 1CNW). (b) Modulation of HCA activity in CLASH-Strep/HCA. A fusion protein of SNAP-tag, a 30-proline linker, NanoLuc Luciferase (NLuc) and HCA and is labelled with a synthetic molecule containing a fluorophore (red star) and the two intramolecular ligands benzenesulfonamide (SA) and biotin (B). Binding of streptavidin (Strep) to the tethered B displaces SA from HCA, leading both to a conformational change that can be seen by a decrease in BRET efficiency, and to an increase of enzymatic activity. (c) Chemical structure of the labelling compound. (d) Enzymatic activity of HCA and followed by measuring absorbance at 348 nm. Streptavidin (Strep) acts as an effector of the hydrolytic activity of HCA: in the absence of Strep, SA binds to and inhibits HCA (black dashed line), while on addition of saturation concentrations of Strep, the catalytic activity increases (black solid line). (e) Emission spectra of 10 nM CLASH-Strep/HCA at increasing streptavidin concentrations. (f) Titration of 10 nM CLASH-Strep/HCA with streptavidin. As a control, the synthetic ligand BG-Cy3-SA lacking the tethered biotin is used.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: Control of HCA by streptavidin.(a) Crystal structure of the active site of HCA bound to a benzenesulfonamide (PDB ID 1CNW). (b) Modulation of HCA activity in CLASH-Strep/HCA. A fusion protein of SNAP-tag, a 30-proline linker, NanoLuc Luciferase (NLuc) and HCA and is labelled with a synthetic molecule containing a fluorophore (red star) and the two intramolecular ligands benzenesulfonamide (SA) and biotin (B). Binding of streptavidin (Strep) to the tethered B displaces SA from HCA, leading both to a conformational change that can be seen by a decrease in BRET efficiency, and to an increase of enzymatic activity. (c) Chemical structure of the labelling compound. (d) Enzymatic activity of HCA and followed by measuring absorbance at 348 nm. Streptavidin (Strep) acts as an effector of the hydrolytic activity of HCA: in the absence of Strep, SA binds to and inhibits HCA (black dashed line), while on addition of saturation concentrations of Strep, the catalytic activity increases (black solid line). (e) Emission spectra of 10 nM CLASH-Strep/HCA at increasing streptavidin concentrations. (f) Titration of 10 nM CLASH-Strep/HCA with streptavidin. As a control, the synthetic ligand BG-Cy3-SA lacking the tethered biotin is used.
Mentions: To demonstrate the generality of CLASH, we applied it to regulate the activity of the enzyme human carbonic anhydrase II (HCA). Furthermore, we wanted to translate the activity modulation into a signal compatible with biosensing. We synthesized a dual ligand in which the HCA inhibitor benzenesulfonamide and a biotin moiety are connected in a way that binding to their respective protein receptors would be mutually exclusive (Fig. 2a–c) and used again streptavidin as the effector. The choice of the linker composition and length in the labelling molecule was based on the crystal structure of the receptor proteins. To couple HCA activity switching to a readout compatible with biosensing, we expressed HCA as a fusion protein with the luciferase NanoLuc, a 30-proline linker (PP30) and SNAP-tag, following the design principles of LUCIDs—our previously described class of BRET-based biosensors18. The SNAP-PP30-NLuc-HCA fusion protein was labelled with the synthetic ligand also containing the fluorophore Cy3 for the BRET readout. The resulting semisynthetic protein CLASH-Strep/HCA (Fig. 2b) is expected to show an increase in HCA activity and display a change in BRET efficiency between NanoLuc and Cy3 when the tethered ligand is displaced from HCA through binding of the effector streptavidin. The interaction between streptavidin and biotin has a Kd in the femtomolar range; since this is much lower than the concentration of CLASH-Strept/HCA in the assay, the transition of the titration curve occurs around the concentration of the CLASH construct.

Bottom Line: We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector.The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector.Tethering such a ligand to the protein of interest results in an intramolecular ligand-protein interaction that can be disrupted through the presence of the effector.

View Article: PubMed Central - PubMed

Affiliation: 1] École Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering, Avenue Forel 2, EPFL SB ISIC LIP BCH-4303, CH-1015 Lausanne, Switzerland [2] École Polytechnique Fédérale de Lausanne, Institute of Bioengineering, CH-1015 Lausanne, Switzerland [3] National Centre of Competence in Research in Chemical Biology, CH-1015 Lausanne, Switzerland.

ABSTRACT
The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand-protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors.

No MeSH data available.


Related in: MedlinePlus