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Experimental determination of the bioluminescence resonance energy transfer (BRET) Forster distances of NanoBRET and red-shifted BRET pairs

ABSTRACT: Bioluminescence Resonance Energy Transfer (BRET) is widely applied to study protein-protein interactions, as well as increasingly to monitor both ligand binding and molecular rearrangements. The Förster distance (R0) describes the physical distance between the two chromophores at which 50% of the maximal energy transfer occurs and it depends on the choice of RET components. R0 can be experimentally determined using flexible peptide linkers of known lengths to separate the two chromophores. Knowledge of the R0 helps to inform on the choice of BRET system. For example, we have previously shown that BRET2 exhibits the largest R0 to date for any genetically encoded RET pair, which may be advantageous for investigating large macromolecular complexes if its issues of low and fast-decaying bioluminescence signal can be accommodated. In this study we have determined R0 for a range of bright and red-shifted BRET pairs, including NanoBRET with tetramethylrhodamine (TMR), non-chloro TOM (NCT), mCherry or Venus as acceptor, and BRET6, a red-shifted BRET2-like system. This study revealed R0 values of 6.15 nm and 6.94 nm for NanoBRET using TMR or NCT as acceptor ligands, respectively. R0 was 5.43 nm for NanoLuc-mCherry, 5.59 nm for NanoLuc-Venus and 5.47 nm for BRET6. This extends the palette of available BRET Förster distances, to give researchers a better-informed choice when considering BRET systems and points towards NanoBRET with NCT as a good alternative to BRET2 as an analysis tool for large macromolecular complexes. Graphical abstract Image 1 Highlights • Experimental determination of Förster distances (R0) for commonly applied BRET pairs.• Determination of R0 for NanoBRET with Venus, mCherry and HaloTag (TMR, NCT).• Determination of R0 for BRET6.• NanoLuc-HaloTag (NCT) exhibits the second largest R0 of any genetically encoded system.

SUBMITTER: Weihs F

PROVIDER: S-EPMC7772631 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Nuclear receptors (NR) regulate transcription of genes involved in many biological processes such as development, cell proliferation, differentiation and cell death. Amongst them, PPARG2 and THR control tissue glucose and lipid homeostasis which are deregulated in severe pathophysiological conditions such as metabolic syndromes. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a real time BRET approach to monitor heterodimerization between RXR and PPARG2 or THR in vitro or in living cells. The presence of a specific DNA target was required to induce in vitro a BRET shift reflecting heterodimerization of RXR/PPARG2 or RXR/THR. As in electrophoretic mobility shift assay (EMSA), the stringency and specificity of the BRET shift assay depended upon assay condition optimization including MgCl2 concentration. For the nuclear receptors, we found by mutagenesis analysis that each heterodimer partner must harbor an intact DNA binding domain to induce BRET and heterodimerization on a DNA target. Moreover the interaction between the PPARG2 ligand binding domain and the RXR DNA binding domain stabilized the heterodimer on its DNA target. BRET microscopy in living cells highlighted the heterodimerization of RXR/PPARG2 within the nucleus clustered in discrete foci that may represent active target gene transcription regulation regions. BRET imaging also suggested that heterodimerization between RXR and PPARG2 required the DNA binding of PPARG2. CONCLUSIONS/SIGNIFICANCE: The BRET approach described here allowed us to study the dynamic interactions which exist between NR in vitro or in living cells and can provide important information on heterodimerization modes, affinity with a given RE and subcellular localization of the heterodimers. This method could be used to study real time changes of NR heterodimers occurring on DNA depending upon cell activation, chromatin state and help to define the mechanisms of ligands or drug action designed to target NRs.

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Experimental determination of the bioluminescence resonance energy transfer (BRET) Forster distances of NanoBRET and red-shifted BRET pairs

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