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• W2040689294 endingPage "e49149" @default.
• W2040689294 startingPage "e49149" @default.
• W2040689294 abstract "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 (pK1/2 = 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." @default.
• W2040689294 created "2016-06-24" @default.
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• W2040689294 date "2012-11-02" @default.
• W2040689294 modified "2023-10-18" @default.
• W2040689294 title "The Single T65S Mutation Generates Brighter Cyan Fluorescent Proteins with Increased Photostability and pH Insensitivity" @default.
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• W2040689294 doi "https://doi.org/10.1371/journal.pone.0049149" @default.
• W2040689294 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3487735" @default.
• W2040689294 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23133673" @default.
• W2040689294 hasPublicationYear "2012" @default.
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