Improving analytical methods for protein-protein interaction through implementation of chemically inducible dimerization.
When investigating interactions between two proteins with complementary reporter tags in yeast two-hybrid or split GFP assays, it remains troublesome to discriminate true- from false-negative results and challenging to compare the level of interaction across experiments. This leads to decreased sensitivity and renders analysis of weak or transient interactions difficult to perform. In this work, we describe the development of reporters that can be chemically induced to dimerize independently of the investigated interactions and thus alleviate these issues. We incorporated our reporters into the widely used split ubiquitin-, bimolecular fluorescence complementation (BiFC)- and Förster resonance energy transfer (FRET)- based methods and investigated different protein-protein interactions in yeast and plants. We demonstrate the functionality of this concept by the analysis of weakly interacting proteins from specialized metabolism in the model plant Arabidopsis thaliana. Our results illustrate that chemically induced dimerization can function as a built-in control for split-based systems that is easily implemented and allows for direct evaluation of functionality.
Center for Dynamic Molecular Interactions (DynaMo), Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Tonni Grube
TG
Andersen
Center for Dynamic Molecular Interactions (DynaMo), Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Sebastian J
SJ
Nintemann
Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Magdalena
M
Marek
Center for Dynamic Molecular Interactions (DynaMo), Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Barbara A
BA
Halkier
Center for Dynamic Molecular Interactions (DynaMo), Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Alexander
A
Schulz
Center for Dynamic Molecular Interactions (DynaMo), Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
Meike
M
Burow
D017360:Arabidopsis / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D019441:Plant Epidermis / Q000166:cytology / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D025941:Protein Interaction Mapping / Q000379:methods*; D055503:Protein Multimerization; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014026:Nicotiana / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
D025801:Ubiquitin