Photobleaching of YFP does not produce a CFP-like species that affects FRET measurements

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Fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent protein (CFP and YFP) fusion proteins assessed as sensitized emission can be effectively controlled by measuring dequenching of CFP fluorescence after YFP photobleaching. Recently Valentin et al.1 reported that photobleaching of YFP induced the formation of a fluorescent product excitable at 405 nm with an emission maximum similar to that of CFP. This could severely affect measurements of FRET between CFP and YFP fusion proteins based on donor dequenching after acceptor photobleaching. Therefore, we have tested whether photoconversion of YFP interferes with CFP-dequenching during photobleaching of YFP at 532 nm in live cells (Supplementary Methods online). Coexpression of non-fused CFP and YFP in Vero cells gave negligible sensitized emission. Correspondingly, photobleaching of YFP did not result in a measurable increase in CFP fluorescence (Fig. 1a). Cells expressing only YFP also did not show 'CFP-like' fluorescence upon YFP photobleaching (Fig. 1b). In contrast, cells expressing a protein in which CFP and YFP were in the same molecule, separated only by 15 amino acids, exhibited a strong donor dequenching effect (Fig. 1c). Coexpression of two interacting SNARE-CFP and -YFP fusion proteins also resulted in rather weak but substantial donor dequenching upon YFP-photobleaching (Fig. 1d). As the reported data1 had been obtained with fixed cells, we performed similar experiments in fixed Vero cells, but photobleaching of YFP again did not result in increased 'CFP-like' fluorescence (Supplementary Fig. 1 online). Photobleaching of YFP-expressing live or fixed cells at 514 instead of 532 nm again did not increase CFP-like fluorescence (Supplementary Fig. 2 online). Finally, spectrofluorimetry with live cells expressing either Venus2 alone or coexpressing Venus and GFP2 fusion proteins, showed also that photobleaching of Venus at 530 nm did not generate a measurable CFP-like product (Supplementary Fig. 3 online). In case of interacting Venus and GFP2 fusion proteins Venus photobleaching was associated with a clear increase of GFP2 fluorescence indicating FRET but without a fluorescence increase in the spectral range of CFP. McAnaney et al.3 reported that at pH 7.2 irradiation of YFP at 514 nm for 10 min led to about 80% photobleaching, which was accompanied by a small increase in fluorescence between 430 and 500 nm during excitation at 390 nm, which in contrast to the data of Valentin et al.1 did not show a distinct maximum at about 470 nm.