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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 a luciferase.(a) Schematic principle of the regulation of a luciferase by an exogenous effector: the dual ligand coelenteramide/biotin can bind to luciferase (Luc) or streptavidin (Strep) separately but not to both at the same time. Binding of streptavidin to the secondary ligand biotin (B) makes the tethered coelenteramide (C) unbind from the luciferase active site, leading to a change in bioluminescence. (b) Crystal structures of the active sites of Renilla luciferase with the inhibitor coelenteramide (blue, PDB ID 2PSJ) and of streptavidin bound to biotin (green, PDB ID 3RY2). (c) Structure of the dual ligand labelling molecule. (d) Luminescence intensity of 100 μl of 25 nM CLASH-Strep/Luc mixed with 2.5 μg ml−1 of the substrate coelenterazine in absence and in presence of 1 μM streptavidin. (e) Picture of the same solutions in transparent tubes, taken with a Canon 600D camera.
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f1: Control of a luciferase.(a) Schematic principle of the regulation of a luciferase by an exogenous effector: the dual ligand coelenteramide/biotin can bind to luciferase (Luc) or streptavidin (Strep) separately but not to both at the same time. Binding of streptavidin to the secondary ligand biotin (B) makes the tethered coelenteramide (C) unbind from the luciferase active site, leading to a change in bioluminescence. (b) Crystal structures of the active sites of Renilla luciferase with the inhibitor coelenteramide (blue, PDB ID 2PSJ) and of streptavidin bound to biotin (green, PDB ID 3RY2). (c) Structure of the dual ligand labelling molecule. (d) Luminescence intensity of 100 μl of 25 nM CLASH-Strep/Luc mixed with 2.5 μg ml−1 of the substrate coelenterazine in absence and in presence of 1 μM streptavidin. (e) Picture of the same solutions in transparent tubes, taken with a Canon 600D camera.

Mentions: Controlling the light-emitting properties of luciferases through molecules of interest is an attractive approach to generate powerful biosensors. Luciferases allosterically controlled by either calcium ions or cyclic AMP have been generated by inserting naturally occurring allosteric protein domains1920, but to the best of our knowledge no luciferases have been designed that are regulated by other proteins. We thus exploited CLASH to transform the luciferase from Renilla reniformis into one that is activated by an effector protein. For first proof-of-principle experiments, we chose the protein streptavidin as the effector, since its natural ligand biotin can be readily synthetically derivatized. We synthesized a dual ligand containing the luciferase inhibitor coelenteramide21 and biotin in such close proximity that simultaneous binding to the luciferase and to streptavidin should be disfavoured (Fig. 1a,b). Using SNAP-tag technology22, we chemically linked the dual ligand to the R. reniformis luciferase, generating CLASH-Strep/Luc (Fig. 1c). In the absence of streptavidin, the tethered coelenteramide inhibits the luciferase, resulting in a dark state. On addition of streptavidin, the light intensity increased in a concentration-dependent manner: binding of streptavidin to the biotin moiety of the synthetic ligand forces coelenteramide to dissociate from the luciferase, leading to an active luciferase. The light intensity increased by 1,700% in the presence of streptavidin, permitting streptavidin detection by naked eye (Fig. 1d,e). The marked increase in light emission in response to an analyte makes the approach an attractive starting point for generating biosensors for various proteins. This then would require the replacement of biotin in our synthetic ligand with an appropriate binder to the protein of interest.


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

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

Control of a luciferase.(a) Schematic principle of the regulation of a luciferase by an exogenous effector: the dual ligand coelenteramide/biotin can bind to luciferase (Luc) or streptavidin (Strep) separately but not to both at the same time. Binding of streptavidin to the secondary ligand biotin (B) makes the tethered coelenteramide (C) unbind from the luciferase active site, leading to a change in bioluminescence. (b) Crystal structures of the active sites of Renilla luciferase with the inhibitor coelenteramide (blue, PDB ID 2PSJ) and of streptavidin bound to biotin (green, PDB ID 3RY2). (c) Structure of the dual ligand labelling molecule. (d) Luminescence intensity of 100 μl of 25 nM CLASH-Strep/Luc mixed with 2.5 μg ml−1 of the substrate coelenterazine in absence and in presence of 1 μM streptavidin. (e) Picture of the same solutions in transparent tubes, taken with a Canon 600D camera.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525150&req=5

f1: Control of a luciferase.(a) Schematic principle of the regulation of a luciferase by an exogenous effector: the dual ligand coelenteramide/biotin can bind to luciferase (Luc) or streptavidin (Strep) separately but not to both at the same time. Binding of streptavidin to the secondary ligand biotin (B) makes the tethered coelenteramide (C) unbind from the luciferase active site, leading to a change in bioluminescence. (b) Crystal structures of the active sites of Renilla luciferase with the inhibitor coelenteramide (blue, PDB ID 2PSJ) and of streptavidin bound to biotin (green, PDB ID 3RY2). (c) Structure of the dual ligand labelling molecule. (d) Luminescence intensity of 100 μl of 25 nM CLASH-Strep/Luc mixed with 2.5 μg ml−1 of the substrate coelenterazine in absence and in presence of 1 μM streptavidin. (e) Picture of the same solutions in transparent tubes, taken with a Canon 600D camera.
Mentions: Controlling the light-emitting properties of luciferases through molecules of interest is an attractive approach to generate powerful biosensors. Luciferases allosterically controlled by either calcium ions or cyclic AMP have been generated by inserting naturally occurring allosteric protein domains1920, but to the best of our knowledge no luciferases have been designed that are regulated by other proteins. We thus exploited CLASH to transform the luciferase from Renilla reniformis into one that is activated by an effector protein. For first proof-of-principle experiments, we chose the protein streptavidin as the effector, since its natural ligand biotin can be readily synthetically derivatized. We synthesized a dual ligand containing the luciferase inhibitor coelenteramide21 and biotin in such close proximity that simultaneous binding to the luciferase and to streptavidin should be disfavoured (Fig. 1a,b). Using SNAP-tag technology22, we chemically linked the dual ligand to the R. reniformis luciferase, generating CLASH-Strep/Luc (Fig. 1c). In the absence of streptavidin, the tethered coelenteramide inhibits the luciferase, resulting in a dark state. On addition of streptavidin, the light intensity increased in a concentration-dependent manner: binding of streptavidin to the biotin moiety of the synthetic ligand forces coelenteramide to dissociate from the luciferase, leading to an active luciferase. The light intensity increased by 1,700% in the presence of streptavidin, permitting streptavidin detection by naked eye (Fig. 1d,e). The marked increase in light emission in response to an analyte makes the approach an attractive starting point for generating biosensors for various proteins. This then would require the replacement of biotin in our synthetic ligand with an appropriate binder to the protein of interest.

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