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Quantitative comparison of different fluorescent protein couples for fast FRET-FLIM acquisition.
Biophys J 2009; 97(8):2368-76BJ

Abstract

The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f(D)) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f(D) percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f(D) value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f(D) (mf(D)) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f(D) analysis.

Authors+Show Affiliations

Institut Jacques Monod, Unite Mixte de Recherche 7592, and Centre National de la Recherche Scientifique, Université Paris-Diderot, Paris, France. sergi_padilla@hotmail.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

19843469

Citation

Padilla-Parra, Sergi, et al. "Quantitative Comparison of Different Fluorescent Protein Couples for Fast FRET-FLIM Acquisition." Biophysical Journal, vol. 97, no. 8, 2009, pp. 2368-76.
Padilla-Parra S, Audugé N, Lalucque H, et al. Quantitative comparison of different fluorescent protein couples for fast FRET-FLIM acquisition. Biophys J. 2009;97(8):2368-76.
Padilla-Parra, S., Audugé, N., Lalucque, H., Mevel, J. C., Coppey-Moisan, M., & Tramier, M. (2009). Quantitative comparison of different fluorescent protein couples for fast FRET-FLIM acquisition. Biophysical Journal, 97(8), pp. 2368-76. doi:10.1016/j.bpj.2009.07.044.
Padilla-Parra S, et al. Quantitative Comparison of Different Fluorescent Protein Couples for Fast FRET-FLIM Acquisition. Biophys J. 2009 Oct 21;97(8):2368-76. PubMed PMID: 19843469.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Quantitative comparison of different fluorescent protein couples for fast FRET-FLIM acquisition. AU - Padilla-Parra,Sergi, AU - Audugé,Nicolas, AU - Lalucque,Hervé, AU - Mevel,Jean-Claude, AU - Coppey-Moisan,Maïté, AU - Tramier,Marc, PY - 2009/05/13/received PY - 2009/06/30/revised PY - 2009/07/21/accepted PY - 2009/10/22/entrez PY - 2009/10/22/pubmed PY - 2010/2/13/medline SP - 2368 EP - 76 JF - Biophysical journal JO - Biophys. J. VL - 97 IS - 8 N2 - The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f(D)) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f(D) percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged in FRET, we tested mTFP1 as a donor coupled to mOrange and EYFP, respectively. mTFP1 turned out to be at least as good as EGFP for donor FRET-FLIM experiments because 1), its lifetime remained constant during light-induced fluorescent changes; 2), its fluorescence decay profile was best fitted with a single exponential model; and 3), no photoconversion was detected. The f(D) value when combined with EYFP as an acceptor was the highest of all tandems tested (0.7). Moreover, in the context of fast acquisitions, we obtained a minimal f(D) (mf(D)) for mTFP1-EYFP that was almost two times greater than that for mCherry-EGFP (0.65 vs. 0.35). Finally, we compared EGFP and mTFP1 in a biological situation in which the fusion proteins were highly immobile, and EGFP and mTFP1 were linked to the histone H4 (EGFP-H4 and mTFP1-H4) in fast FLIM acquisitions. In this particular case, the fluorescence intensity was more stable for EGFP-H4 than for mTFP1-H4. Nevertheless, we show that mTFP1/EYFP stands alone as the best FRET-FLIM couple in terms of f(D) analysis. SN - 1542-0086 UR - https://www.unboundmedicine.com/medline/citation/19843469/Quantitative_comparison_of_different_fluorescent_protein_couples_for_fast_FRET_FLIM_acquisition_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0006-3495(09)01307-1 DB - PRIME DP - Unbound Medicine ER -