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PLoS One. 2012;7(11):e49149. doi: 10.1371/journal.pone.0049149. Epub 2012 Nov 2.

The single T65S mutation generates brighter cyan fluorescent proteins with increased photostability and pH insensitivity.

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  • 1Laboratoire de Chimie Physique, CNRS, Université Paris Sud, Orsay, France.


Cyan fluorescent proteins (CFP) derived from Aequorea victoria GFP, carrying a tryptophan-based chromophore, are widely used as FRET donors in live cell fluorescence imaging experiments. Recently, several CFP variants with near-ultimate photophysical performances were obtained through a mix of site-directed and large scale random mutagenesis. To understand the structural bases of these improvements, we have studied more specifically the consequences of the single-site T65S mutation. We find that all CFP variants carrying the T65S mutation not only display an increased fluorescence quantum yield and a simpler fluorescence emission decay, but also show an improved pH stability and strongly reduced reversible photoswitching reactions. Most prominently, the Cerulean-T65S variant reaches performances nearly equivalent to those of mTurquoise, with QY  = 0.84, an almost pure single exponential fluorescence decay and an outstanding stability in the acid pH range (pK(1/2) = 3.6). From the detailed examination of crystallographic structures of different CFPs and GFPs, we conclude that these improvements stem from a shift in the thermodynamic balance between two well defined configurations of the residue 65 hydroxyl. These two configurations differ in their relative stabilization of a rigid chromophore, as well as in relaying the effects of Glu222 protonation at acid pHs. Our results suggest a simple method to greatly improve numerous FRET reporters used in cell imaging, and bring novel insights into the general structure-photophysics relationships of fluorescent proteins.

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