Techniques


BRET - Bioluminescence Resonance Energy Transfer

Schematische Darstellung eines BRET-Experiments.
Schematische Darstellung eines BRET-Experiments: Expression der rekombinanten BRET-Konstrukte in 96-Well-Platten und anschließende Analyse im α-Fusion™ Lesegerät mit zugehöriger Auswertung.

We recently developed a Bioluminescence Resonance Energy Transfer (BRET)-based assay to investigate protein-protein interactions of PKA regulatory (RI/RII) and catalytic (Cα/PrKX) subunits in living mammalian cells (Diskar et al., 2005; 2006; 2007; Prinz et al., 2004; 2006a; 2006b). It is based on the physical principle of resonance energy transfer, which was first described by Förster in 1948 (Förster, 1948).

BRET is a proximity assay based on the detection of energy transfer between Renilla luciferase (RLuc) used as the donor protein and a variant of Green Fluorescent Protein (GFP, here: GFP²) as the acceptor. Upon oxidation of the substrate Deep Blue C, a coelenterazine derivative, RLuc emitts light at 410 nm. If GFP² is in close proximity to the luciferase, part of the light will be transferred by resonance to GFP², and, in turn, GFP² will emitt light at 515 nm. Both proteins have been genetically modified and optimized for the use this assay and for high expression results in eukaryote cell culture.

We have designed and cloned various vectors expressing recombinant proteins using the BRET system, by fusing the coding sequences of mainly human RI-, RII and C- subunits of PKA to the N- and C- terminus of either GFP² or Rluc.
When RLuc and GFP² are expressed as fusion proteins in a cell, the interaction event of the target proteins (here: RI/RII- and C- subunits of PKA) can be measured by the signal ratio of green over blue light (515/410nm) using an α-Fusion™ multilabel reader.

An advantage of BRET over the earlier developed FRET (Fluorescence Resonance Energy Transfer) is that BRET does not require excitation by an external light source, thus eliminating photo bleaching and high fluorescent background problems in the cell-based assay (Prinz et al., 2008).

We successfully established a medium throughput assay system in 96-well microplate format using the BRET-technology suitable for probing cAMP analogs affecting the activity of PKA by enhancing or attenuating the dissociation of PKA holoenzyme (Gesellchen et al., 2006; Moll et al., 2006; Prinz et al., 2006a; 2006b). The assay was also utilized for detecting anchoring dependent signalling propagation mediated by A-kinase anchoring proteins (Hundsrucker et al., 2006; Prinz et al., 2006a) and isoform specific regulation of PKA (Diskar et al., 2007).

More literature:
- Förster, T. (1948) Zwischenmolekulare Energiewanderung und Fluoreszenz. Ann. Physik 2, 55-75.

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FP - Fluorescence polarisation

Modell von Fluorescein markiertem cAMP in der Bindungstasche der regulatorischen Untereinheit der cAMP-abhängigen Proteinkinase A.
Modell von Fluorescein markiertem cAMP in der Bindungstasche der regulatorischen Untereinheit der cAMP-abhängigen Proteinkinase A.

In biological and medical research the essential aspect of fluorescence polarisation (FP) is, that FP provides information on the rotational mobility of a fluorescently labelled molecule. Since the mobility of the fluorescent molecule changes upon binding to a large macromolecule, one can utilize FP to determine and quantify biomolecular interactions. Consequently, fluorescence polarization has been used now for many years to study biochemical systems, for example protein-protein and protein-ligand interactions (Jameson et al., 2003; Moll et al., 2006c). The advantages of this method are that the assay is inexpensive (low sample amount needed) and rapid with a sensitivity close to classical radioligand binding assays in a homogenous high throughput format. The theoretical principles of FP (or fluorescence anisotropy, which are both describing the same physical phenomenon) have been extensively described (Lakowicz, 1999; Moll et al., 2006a, 2006b; 2006c).

Since 2006 we establish in our group direct and indirect FP assay formats to basically determine the binding of labelled and unlabeld cyclic nucleotides (e.g. cAMP and derivatives) to cyclic nucleotide binding proteins and domains thereof (Moll et al., 2006a).

So far we therefore measure the affinity of hundreds of cyclic nucleotides to cAMP dependent protein kinase (Moll et al., 2006a; 2008; Schweinsberg et al., 2008), Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (Lolicato et al., 2011) and cGMP-dependent protein kinase.

More literature:
- Jameson, D.M. and Croney, J.C. (2003). "Fluorescence polarization: past, present and future." Comb Chem High Throughput Screen 6(3): 167-173.
- Lakowicz, J.R. (1999). "Fluorescence Anisotropy. Principles of fluorescence Spectroscopy." New York, Kluwer academic/Plenum Publishers: 291-319.

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