Engineering fluorescent proteins towards ultimate performances: lessons from the newly developed cyan variants

New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to intensive engineering based on random mutagenesis and directed evolution. However, most of these newly developed FPs fail to achieve all the performances required for their bioimaging applications. The design of highly optimised FP-based reporters, simultaneously displaying appropriate colour, multimeric state, chromophore maturation, brightness, photostability and environmental sensitivity will require a better understanding of the structural and dynamic determinants of FP photophysics.The recent development of cyan fluorescent proteins (CFPs) like mCerulean3, mTurquoise2 and Aquamarine brings a different view on these questions, as in this particular case, a step by step evaluation of critical mutations has been performed within a family of spectrally identical and evolutionary close variants. These efforts have led to CFPs with quantum yields close to unity, near single exponential emission decays, high photostability and complete insensitivity to pH, making them ideal choices as energy transfer donors in FRET and FLIM imaging applications. During this process, it was found that a proper amino-acid choice at only two positions (148 and 65) is sufficient to transform the performances of CFPs: with the help of structural and theoretical investigations, we rationalise here how these two positions critically control the CFP photophysics, in the context of FPs derived from theAequorea victoriaspecies.Today, these results provide a useful toolbox for upgrading the different CFP donors carried by FRET biosensors. They also trace the route towards thede novodesign of FP-based optogenetic devices that will be perfectly tailored to dedicated imaging and sensing applications.