The single T65S mutation generates brighter cyan fluorescent proteins with increased photostability and pH insensitivity.
PLoS One
; 7(11): e49149, 2012.
Article
in En
| MEDLINE
| ID: mdl-23133673
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 â=â0.84, an almost pure single exponential fluorescence decay and an outstanding stability in the acid pH range (pK(1/2)â=â3.6). 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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Photochemistry
/
Green Fluorescent Proteins
/
Mutation
Type of study:
Prognostic_studies
Limits:
Animals
Language:
En
Journal:
PLoS One
Journal subject:
CIENCIA
/
MEDICINA
Year:
2012
Document type:
Article
Affiliation country:
France