Abstract
Enzyme–substrate interactions are weak and occur only transiently and thus, a faithful analysis of these interactions typically requires elaborated biochemical methodology. The bimolecular-fluorescence complementation (BiFC) assay, also referred to as split YFP assay, is a powerful and straightforward tool to test protein–protein interactions. This system is commonly used due to many advantages and especially due to its simple ease of use. BIFC relies on the reconstitution of an N-terminal and C-terminal half of YFP into a functional, i.e., fluorescent protein. Noteworthy, the dissociation constant of the two YFP halves is much lower than the association constant leading to a stabilization of the protein–protein interaction to be monitored. Whereas this property is sometimes critical, it also increases the sensitivity of the detection system by stabilizing transient interactions. Here, we exploit this property to detect and monitor interaction between a kinase and its substrate. In particular, we characterize with the BiFC system kinase-variants that show an altered substrate binding.
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Acknowledgments
We thank Annika K. Weimer for critical reading and comments on the manuscript and Klaus Harter and Jane Parker for sharing the plasmids. N.D. is a fellow of the International Max Planck Research School and funded by the Max Planck Society, work in the laboratory of A.S. is supported through an ATIP grant from the Centre National de la Recherche Scientifique (CNRS) and an ERC starting grant from the European Union.
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Pusch, S., Dissmeyer, N., Schnittger, A. (2011). Bimolecular-Fluorescence Complementation Assay to Monitor Kinase–Substrate Interactions In Vivo. In: Dissmeyer, N., Schnittger, A. (eds) Plant Kinases. Methods in Molecular Biology, vol 779. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-61779-264-9_14
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DOI: https://doi.org/10.1007/978-1-61779-264-9_14
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