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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

Modulation of HCA by tacrine on living cells.(a) Cartoon of CLASH-AChE/HCA. The acetylcholine esterase (AChE) inhibitor tacrine (T) displaces edrophonium (E) from AChE and benzenesulfonamide (SA) can bind to HCA inducing an increase in FRET efficiency. (b) Structure of the labelling compound. (c) Fluorescence micrographs of HEK293 cells displaying CLASH-AChE/HCA on their surface. In the absence of tacrine Cy3 emission is more intense, while on addition tacrine Cy5 emission increases; scale bars, 50 μm. (d) Changes observed in FRET ratio, donor (Cy3) and acceptor (Cy5) emission on perfusion of HEK293 cells displaying CLASH-AChE/HCA on their surface with 3 mM tacrine.
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f4: Modulation of HCA by tacrine on living cells.(a) Cartoon of CLASH-AChE/HCA. The acetylcholine esterase (AChE) inhibitor tacrine (T) displaces edrophonium (E) from AChE and benzenesulfonamide (SA) can bind to HCA inducing an increase in FRET efficiency. (b) Structure of the labelling compound. (c) Fluorescence micrographs of HEK293 cells displaying CLASH-AChE/HCA on their surface. In the absence of tacrine Cy3 emission is more intense, while on addition tacrine Cy5 emission increases; scale bars, 50 μm. (d) Changes observed in FRET ratio, donor (Cy3) and acceptor (Cy5) emission on perfusion of HEK293 cells displaying CLASH-AChE/HCA on their surface with 3 mM tacrine.

Mentions: To create CLASH-AChE/HCA, AChE was fused to the N-terminus of an HCA-based SNIFIT (Fig. 4a). We targeted the construct to the outer cell membrane of transiently transfected HEK293 cells. The cells were labelled with a dual ligand containing benzenesulfonamide as primary ligand and edrophonium, an inhibitor of AChE26, as secondary ligand (Fig. 4b). In the absence of free AChE inhibitors, the stronger interaction of edrophonium with AChE prevents the binding of the primary ligand to HCA, keeping the sensor in its low-FRET state. When the AChE inhibitor tacrine, a drug used in the past to treat Alzheimer's disease, was perfused on living cells expressing CLASH-AChE/HCA, tacrine displaced the secondary ligand from AChE, allowing the benzenesulfonamide to bind HCA and leading to high FRET efficiency of the sensor (Fig. 4c-d). Importantly, the switching between the two states was fully reversible, and the overall measured FRET ratio change was twofold higher than the original ACh-SNIFIT26. In CLASH-AChE/HCA, the relative strength of the two synthetic ligands is such that HCA is active in the absence of the effector targeting AChE and inhibited by its addition (see also Supplementary Discussion for a more detailed discussion of the underlying design principles). Choosing a stronger HCA ligand, it should be equally possible to generate a variant, namely CLASH-HCA/AChE, in which AChE activity is controlled by small benzenesulfonamides. As AChE is a key enzyme for neurotransmission both at the central and at the peripheral nervous systems, such a CLASH-HCA/AChE could find applications as a research tool in neurobiology. For example, inactivating its AChE activity by the addition of a biorthogonal small molecule, for example, a sulfonamide, could help in elucidating the role of cholinergic pathways in neurobiology.


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

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

Modulation of HCA by tacrine on living cells.(a) Cartoon of CLASH-AChE/HCA. The acetylcholine esterase (AChE) inhibitor tacrine (T) displaces edrophonium (E) from AChE and benzenesulfonamide (SA) can bind to HCA inducing an increase in FRET efficiency. (b) Structure of the labelling compound. (c) Fluorescence micrographs of HEK293 cells displaying CLASH-AChE/HCA on their surface. In the absence of tacrine Cy3 emission is more intense, while on addition tacrine Cy5 emission increases; scale bars, 50 μm. (d) Changes observed in FRET ratio, donor (Cy3) and acceptor (Cy5) emission on perfusion of HEK293 cells displaying CLASH-AChE/HCA on their surface with 3 mM tacrine.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525150&req=5

f4: Modulation of HCA by tacrine on living cells.(a) Cartoon of CLASH-AChE/HCA. The acetylcholine esterase (AChE) inhibitor tacrine (T) displaces edrophonium (E) from AChE and benzenesulfonamide (SA) can bind to HCA inducing an increase in FRET efficiency. (b) Structure of the labelling compound. (c) Fluorescence micrographs of HEK293 cells displaying CLASH-AChE/HCA on their surface. In the absence of tacrine Cy3 emission is more intense, while on addition tacrine Cy5 emission increases; scale bars, 50 μm. (d) Changes observed in FRET ratio, donor (Cy3) and acceptor (Cy5) emission on perfusion of HEK293 cells displaying CLASH-AChE/HCA on their surface with 3 mM tacrine.
Mentions: To create CLASH-AChE/HCA, AChE was fused to the N-terminus of an HCA-based SNIFIT (Fig. 4a). We targeted the construct to the outer cell membrane of transiently transfected HEK293 cells. The cells were labelled with a dual ligand containing benzenesulfonamide as primary ligand and edrophonium, an inhibitor of AChE26, as secondary ligand (Fig. 4b). In the absence of free AChE inhibitors, the stronger interaction of edrophonium with AChE prevents the binding of the primary ligand to HCA, keeping the sensor in its low-FRET state. When the AChE inhibitor tacrine, a drug used in the past to treat Alzheimer's disease, was perfused on living cells expressing CLASH-AChE/HCA, tacrine displaced the secondary ligand from AChE, allowing the benzenesulfonamide to bind HCA and leading to high FRET efficiency of the sensor (Fig. 4c-d). Importantly, the switching between the two states was fully reversible, and the overall measured FRET ratio change was twofold higher than the original ACh-SNIFIT26. In CLASH-AChE/HCA, the relative strength of the two synthetic ligands is such that HCA is active in the absence of the effector targeting AChE and inhibited by its addition (see also Supplementary Discussion for a more detailed discussion of the underlying design principles). Choosing a stronger HCA ligand, it should be equally possible to generate a variant, namely CLASH-HCA/AChE, in which AChE activity is controlled by small benzenesulfonamides. As AChE is a key enzyme for neurotransmission both at the central and at the peripheral nervous systems, such a CLASH-HCA/AChE could find applications as a research tool in neurobiology. For example, inactivating its AChE activity by the addition of a biorthogonal small molecule, for example, a sulfonamide, could help in elucidating the role of cholinergic pathways in neurobiology.

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