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Optimizing fluorescent protein trios for 3-Way FRET imaging of protein interactions in living cells.

ABSTRACT: Powerful new methods have extended FRET microscopy to the imaging of three or more interacting proteins inside living cells. Here, we compared widely available fluorescent proteins to find the best trio for 3-Way FRET imaging. We focused on readily available cyan, yellow, and red proteins that have high quantum yields, large extinction coefficients and good photostability, which defined these candidate proteins: CyPet/mTFP1/mTurqoise2, mCitrine/YPet, and TagRFP/TagRFPt/mRuby2/mCherry. By taking advantage of the high structural similarity across the fluorescent proteins, we generated structurally similar, but photophysically distinct donor/acceptor and triple fluorophore fusion proteins and measured their FRET efficiencies inside living cells. Surprisingly, their published photophysical parameters and calculated Förster distances did not predict the best combinations of FPs. Using cycloheximide to inhibit protein synthesis, we found that the different FP maturation rates had a strong effect on the FRET efficiency. This effect was pronounced when comparing rapidly maturing yellow and slowly maturing red FPs. We found that red FPs with inferior photophysics gave superior FRET efficiencies because of faster maturation rates. Based on combined metrics for the FRET efficiency, fluorophore photophysics and fluorophore maturation we determined that Turqoise2, YPet and Cherry were the best available FPs for live cell 3-Way FRET measurements.

SUBMITTER: Scott BL

PROVIDER: S-EPMC4487001 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Optimizing fluorescent protein trios for 3-Way FRET imaging of protein interactions in living cells.

Scott Brandon L BL Hoppe Adam D AD

Scientific reports 20150701

Powerful new methods have extended FRET microscopy to the imaging of three or more interacting proteins inside living cells. Here, we compared widely available fluorescent proteins to find the best trio for 3-Way FRET imaging. We focused on readily available cyan, yellow, and red proteins that have high quantum yields, large extinction coefficients and good photostability, which defined these candidate proteins: CyPet/mTFP1/mTurqoise2, mCitrine/YPet, and TagRFP/TagRFPt/mRuby2/mCherry. By taking ...[more]

PMID: 26130463

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Project description:PPAR? is a pharmacological target in inflammatory and metabolic diseases. Upon agonistic treatment or following antagonism, binding of co-factors is altered, which consequently affects PPAR?-dependent transactivation as well as its DNA-independent properties. Therefore, establishing techniques to characterize these interactions is an important issue in living cells. Methods: Using the FRET pair Clover/mRuby2, we set up a flow cytometry-based FRET assay by analyzing PPAR?1 binding to its heterodimerization partner RXR?. Analyses of PPAR?-reporter and co-localization studies by laser-scanning microscopy validated this system. Refining the system, we created a new readout to distinguish strong from weak interactions, focusing on PPAR?-binding to the co-repressor N-CoR2. Results: We observed high FRET in cells expressing Clover-PPAR?1 and mRuby2-RXR?, but no FRET when cells express a mRuby2-RXR? deletion mutant, lacking the PPAR? interaction domain. Focusing on the co-repressor N-CoR2, we identified in HEK293T cells the new splice variant N-CoR2-?ID1-exon. Overexpressing this isoform tagged with mRuby2, revealed no binding to Clover-PPAR?1, nor in murine J774A.1 macrophages. In HEK293T cells, binding was even lower in comparison to N-CoR2 constructs in which domains established to mediate interaction with PPAR? binding are deleted. These data suggest a possible role of N-CoR2-?ID1-exon as a dominant negative variant. Because binding to N-CoR2-mRuby2 was not altered following activation or antagonism of Clover-PPAR?1, we determined the effect of pharmacological treatment on FRET intensity. Therefore, we calculated flow cytometry-based FRET efficiencies based on our flow cytometry data. As with PPAR? antagonism, PPAR? agonist treatment did not prevent binding of N-CoR2. Conclusion: Our system allows the close determination of protein-protein interactions with a special focus on binding intensity, allowing this system to characterize the role of protein domains as well as the effect of pharmacological agents on protein-protein interactions.

Dataset Information

Optimizing fluorescent protein trios for 3-Way FRET imaging of protein interactions in living cells.

Publications

Optimizing fluorescent protein trios for 3-Way FRET imaging of protein interactions in living cells.

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