Enhanced yellow fluorescent protein photoconversion to a cyan fluorescent protein-like species is sensitive to thermal and diffusion conditions

Ongoing research efforts into fluorescent proteins continuously generates new mutation variants, some of which can become photoactivated or photoconverted to a red-shifted color upon intense UV or blue light illumination. We report a built-in propensity for enhanced yellow fluorescent protein (EYFP) to undergo irreversible photoconversion into a cyan fluorescent protein (CFP)-like species upon green-light illumination. The photoconversion is thermally activated, happens mainly in fixed, nonsealed cell samples, and may result in a very bright and relatively photostable CFP-like species. The photoconversion efficiency depends on the sample diffusivity and is much increased in dehydrated, oxygenated samples. Given the large variations in conversion efficiency observed among samples as well as within a sample, photoconversion cannot be appropriately accounted for in the analysis of acceptor photobleaching fluorescence resonance energy transfer (pbFRET) images and should rather be completely avoided. Thus, samples should always be checked and discarded if photoconversion is observed.