Combining chemical and genetic approaches for development of responsive FRET-based sensor systems for protein kinases

- Steady-state and time-gated FRET sensor systems were developed based on singlet-singlet and triplet-singlet energy transfers.
- Triplet-singlet energy transfer between selenium-comprising heteroaromatic fragment of ARC-Lum(−) probe and TagRFP-fused PIM2
- Transport peptide nona(d-arginine) amide binds to PIM2 kinase with high affinity.
- Time-gated FRET assay using Lumi4Tb-labeled ARC probes and TagRFP-fused PIM2
- PIM2 concentration in cell lysate was determined using ARC-Lum probes.

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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