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Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule.

Cho U, Zimmerman SM, Chen LC, Owen E, Kim JV, Kim SK, Wandless TJ - PLoS ONE (2013)

Bottom Line: To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C.We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans.These data reinforce that DD can function in virtually any organism and temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.

ABSTRACT
Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

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Related in: MedlinePlus

Schematic showing that the stability of a protein of interest (POI) fused to a destabilizing domain (DD) can be controlled using a high-affinity, stabilizing ligand.
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pone-0072393-g001: Schematic showing that the stability of a protein of interest (POI) fused to a destabilizing domain (DD) can be controlled using a high-affinity, stabilizing ligand.

Mentions: One of the fundamental experimental strategies of modern molecular biology is “perturb and observe”. Investigators perturb a complex biological system and, by observing the consequences of their experimental change, infer the function of the perturbed component. Perturbations that are specific (i.e., only modulate the molecule under investigation) are desired in order for researchers to be confident in their conclusions. To this end, we developed a small, metastable protein domain whose metabolic stability relies on the presence of a high-affinity, small molecule ligand [1]. These domains, which we call destabilizing domains (DDs), can be genetically fused to any protein of interest, resulting in a fusion protein that, when expressed in eukaryotic cells, is constitutively degraded by the 26S proteasome in the absence of the stabilizing ligand. However, the addition of a cell-permeable, high-affinity ligand stabilizes the DD in a rapid, dose-dependent, and reversible manner (Figure 1).


Rapid and tunable control of protein stability in Caenorhabditis elegans using a small molecule.

Cho U, Zimmerman SM, Chen LC, Owen E, Kim JV, Kim SK, Wandless TJ - PLoS ONE (2013)

Schematic showing that the stability of a protein of interest (POI) fused to a destabilizing domain (DD) can be controlled using a high-affinity, stabilizing ligand.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750007&req=5

pone-0072393-g001: Schematic showing that the stability of a protein of interest (POI) fused to a destabilizing domain (DD) can be controlled using a high-affinity, stabilizing ligand.
Mentions: One of the fundamental experimental strategies of modern molecular biology is “perturb and observe”. Investigators perturb a complex biological system and, by observing the consequences of their experimental change, infer the function of the perturbed component. Perturbations that are specific (i.e., only modulate the molecule under investigation) are desired in order for researchers to be confident in their conclusions. To this end, we developed a small, metastable protein domain whose metabolic stability relies on the presence of a high-affinity, small molecule ligand [1]. These domains, which we call destabilizing domains (DDs), can be genetically fused to any protein of interest, resulting in a fusion protein that, when expressed in eukaryotic cells, is constitutively degraded by the 26S proteasome in the absence of the stabilizing ligand. However, the addition of a cell-permeable, high-affinity ligand stabilizes the DD in a rapid, dose-dependent, and reversible manner (Figure 1).

Bottom Line: To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C.We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans.These data reinforce that DD can function in virtually any organism and temperature.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.

ABSTRACT
Destabilizing domains are conditionally unstable protein domains that can be fused to a protein of interest resulting in degradation of the fusion protein in the absence of stabilizing ligand. These engineered protein domains enable rapid, reversible and dose-dependent control of protein expression levels in cultured cells and in vivo. To broaden the scope of this technology, we have engineered new destabilizing domains that perform well at temperatures of 20-25°C. This raises the possibility that our technology could be adapted for use at any temperature. We further show that these new destabilizing domains can be used to regulate protein concentrations in C. elegans. These data reinforce that DD can function in virtually any organism and temperature.

Show MeSH
Related in: MedlinePlus