title: The single T65S mutation generates brighter cyan fluorescent proteins with increased photostability and pH insensitivity.
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 (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.
authors.affiliation: Laboratoire de Chimie Physique, CNRS, Université Paris Sud, Orsay, France.
authors.forename: Asma
authors.initials: A
authors.lastname: Fredj
authors.forename: Hélène
authors.initials: H
authors.lastname: Pasquier
authors.forename: Isabelle
authors.initials: I
authors.lastname: Demachy
authors.forename: Gabriella
authors.initials: G
authors.lastname: Jonasson
authors.forename: Bernard
authors.initials: B
authors.lastname: Levy
authors.forename: Valérie
authors.initials: V
authors.lastname: Derrien
authors.forename: Yasmina
authors.initials: Y
authors.lastname: Bousmah
authors.forename: Gallia
authors.initials: G
authors.lastname: Manoussaris
authors.forename: Frank
authors.initials: F
authors.lastname: Wien
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Ridard
authors.forename: Marie
authors.initials: M
authors.lastname: Erard
authors.forename: Fabienne
authors.initials: F
authors.lastname: Merola
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D004285:Dogs; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010777:Photochemistry / Q000379:methods*; D010825:Physics / Q000379:methods; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D017356:Synchrotrons; D013997:Time Factors
chemical_list: C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins