An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching

Abstract

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

Biocompatibility of a genetically encoded calcium indicator in a transgenic mouse model

“…This reduces calcium buffering caused by the indicator and potential residual biological activity of the sensing domain. Other improvements may arise from incorporating brighter fluorescent protein variants, such as mCerulean3 45 or mTurquoise 46 , to allow lower expression levels while maintaining the same signal-to-noise levels.…”

Future Perspective of Single-Molecule FRET Biosensors and Intravital FRET Microscopy

“…Recently, CFP mutants with a single, long lifetime and increased brightness and photostability (e.g., mTurquoise or mCerulean3) have been developed that are suitable for donor FPs in FRET biosensors, with great potential for in vivo application (48,49). However, there is a paradoxical concern about the application of this pair to two-photon excitation intravital FRET microscopy, mainly due to the proximity of their excitation profiles at relatively short wavelength.…”

A simple approach for measuring FRET in fluorescent biosensors using two-photon microscopy

“…Since the cyan FPs (CFP) are optimally excited close to the power maximum of the titanium-sapphire lasers used in most TPLSM systems, we focused our evaluations of potential FRET donors on the newer variants of the CFPs that have improved brightness and photostability, and no photo-switching behavior [34][35][36][37] . Based on previous studies demonstrating that wavelengths near 800 nm could be used for relatively selective excitation of CFP over YFP 38,39 , we focused our evaluations of potential FRET acceptors on newer, improved variants of YFP.…”