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A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex
Published on: April 11, 2017
Combining chemical and genetic approaches for development of responsive FRET-based sensor systems for protein kinases
Ganesh Babu Manoharan1, Erki Enkvist1, Asko Uri1
1Institute of Chemistry, University of Tartu, Ravila 14A, 50411 Tartu, Estonia.
Abstract:
Chemical and genetic approaches were combined for the development of responsive FRET-based sensor systems for protein kinases, using PIM2 as the model kinase. Fusions of PIM2 and a red fluorescent protein, TagRFP were expressed in mammalian cells and small-molecule ARC-Lum photoluminescent probes possessing different phosphorescent and fluorescent properties were constructed. Based on a variety of Förster-type resonant energy transfer (FRET) mechanisms (including intermolecular or intramolecular energy transfer and transfer between singlet-singlet or triplet-singlet electronic states of interacting luminophores) of the probe and that of the fluorescently tagged PIM2, FRET-based sensor systems were constructed. The developed assays can be applied for analysis of PIM2 in biological samples and screening and characterization of PIM2 inhibitors in cell lysates. In screening studies sub-micromolar affinity of a d-arginine-rich peptide, nona(d-arginine) amide [(d-Arg)9-NH2], towards PIM2 was discovered that points to possible specific effect of this widely used transport peptide to cellular protein phosphorylation balance.
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