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 (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.

Quantification of Förster resonance energy transfer by monitoring sensitized emission in living plant cells

“…Cerulean3 shows high photostability and reduced photoswitching behavior, a high quantum yield of 0.87, and only a slightly reduced absorption coefficient compared to Cerulean. Reversing only S65T stabilized the H-bonding status of the hydroxyl group and resulted in a Cerulean with increased photostability and pH-resistance, high quantum yield, and reduced reversible photoswitching (Fredj et al, 2012). …”

Rational design of a pH‐insensitive cyan fluorescent protein CyPet2 based on the CyPet crystal structure

“…We first determined the emission spectra of CyPet under different pH conditions. We found that the emission spectra of CyPet at pH 5.2 exhibited double emission peaks at 464 nm and 480 nm as some other CFPs [10,14]. Under conditions of neutral pH (pH 7.0), we show that this double emission peak converts to a single emission peak, positioned at 474 nm.…”

“…We found that the emission spectra of CyPet at pH 5.2 exhibited double emission peaks at 464 nm and 480 nm as some other CFPs [10,14]. We found that the emission spectra of CyPet at pH 5.2 exhibited double emission peaks at 464 nm and 480 nm as some other CFPs [10,14].…”

Identification of a Fluorescent Protein from Rhacostoma Atlantica

“…The homogeneous lifetime decay seen for the Rhacostoma FP may be desirable for fluorescence‐lifetime imaging microscopy (FLIM)‐based energy transfer studies. Much effort has been placed on improving the CFP for energy transfer studies, part of which was the development of homogeneous lifetime decays , although the shapes of the emission spectra were essentially unchanged. Another potential energy donor to the YFP is the teal FP (TFP) , having excitation and emission peaks close to the Rhacostoma FP peaks.…”