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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.


Modulation of HCA by DHFR and DHFR ligands.Cartoon of CLASH-DHFR/HCA for control of HCA with DHFR: DHFR acts as an effector by binding to the tethered trimethoprim (T) and leading to the unbinding of benzenesulfonamide (SA) from HCA. (b) Chemical structure of the labelling molecule. (c) The opening of the biosensor can be detected by the increase in emission ratio on addition of DHFR. (d) Titrations with the anticancer agent methotrexate (MTX) at three different free DHFR concentrations show that the colour of the luminescence emission changes at the threshold concentration. Picture taken with a Canon 600D camera. The colour balance was modified to give a clear difference between the blue and red colours.
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f3: Modulation of HCA by DHFR and DHFR ligands.Cartoon of CLASH-DHFR/HCA for control of HCA with DHFR: DHFR acts as an effector by binding to the tethered trimethoprim (T) and leading to the unbinding of benzenesulfonamide (SA) from HCA. (b) Chemical structure of the labelling molecule. (c) The opening of the biosensor can be detected by the increase in emission ratio on addition of DHFR. (d) Titrations with the anticancer agent methotrexate (MTX) at three different free DHFR concentrations show that the colour of the luminescence emission changes at the threshold concentration. Picture taken with a Canon 600D camera. The colour balance was modified to give a clear difference between the blue and red colours.

Mentions: To demonstrate the modularity of our approach, we took the same HCA fusion protein as above, but used the enzyme dihydrofolate reductase (DHFR) as the effector. Towards this end, we labelled SNAP-PP30-NLuc-HCA with a synthetic ligand that consists of the DHFR-ligand trimethoprim and benzenesulfonamide, generating CLASH-DHFR/HCA (Fig. 3a,b). In the absence of DHFR, the benzenesulfonamide occupies the active site of HCA and CLASH-DHFR/HCA is in a closed conformation, as shown by the high BRET efficiency (Fig. 3c). In the presence of sufficient concentrations of DHFR, the binding of the tethered sulfonamide to the active site of HCA is prevented and CLASH-DHFR/HCA adopts an open conformation, as demonstrated by its low BRET efficiency.


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

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

Modulation of HCA by DHFR and DHFR ligands.Cartoon of CLASH-DHFR/HCA for control of HCA with DHFR: DHFR acts as an effector by binding to the tethered trimethoprim (T) and leading to the unbinding of benzenesulfonamide (SA) from HCA. (b) Chemical structure of the labelling molecule. (c) The opening of the biosensor can be detected by the increase in emission ratio on addition of DHFR. (d) Titrations with the anticancer agent methotrexate (MTX) at three different free DHFR concentrations show that the colour of the luminescence emission changes at the threshold concentration. Picture taken with a Canon 600D camera. The colour balance was modified to give a clear difference between the blue and red colours.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Modulation of HCA by DHFR and DHFR ligands.Cartoon of CLASH-DHFR/HCA for control of HCA with DHFR: DHFR acts as an effector by binding to the tethered trimethoprim (T) and leading to the unbinding of benzenesulfonamide (SA) from HCA. (b) Chemical structure of the labelling molecule. (c) The opening of the biosensor can be detected by the increase in emission ratio on addition of DHFR. (d) Titrations with the anticancer agent methotrexate (MTX) at three different free DHFR concentrations show that the colour of the luminescence emission changes at the threshold concentration. Picture taken with a Canon 600D camera. The colour balance was modified to give a clear difference between the blue and red colours.
Mentions: To demonstrate the modularity of our approach, we took the same HCA fusion protein as above, but used the enzyme dihydrofolate reductase (DHFR) as the effector. Towards this end, we labelled SNAP-PP30-NLuc-HCA with a synthetic ligand that consists of the DHFR-ligand trimethoprim and benzenesulfonamide, generating CLASH-DHFR/HCA (Fig. 3a,b). In the absence of DHFR, the benzenesulfonamide occupies the active site of HCA and CLASH-DHFR/HCA is in a closed conformation, as shown by the high BRET efficiency (Fig. 3c). In the presence of sufficient concentrations of DHFR, the binding of the tethered sulfonamide to the active site of HCA is prevented and CLASH-DHFR/HCA adopts an open conformation, as demonstrated by its low BRET efficiency.

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.