Bimodal Detection of Proteins by 129 Xe NMR and Fluorescence Spectroscopy.

A full understanding of biological phenomena involves sensitive and noninvasive detection. Herein, we report the optimization of a probe for intracellular proteins that combines the advantages of fluorescence and hyperpolarized 129 Xe NMR spectroscopy detection. The fluorescence detection part is composed of six residues containing a tetracysteine tag (-CCXXCC-) genetically incorporated into the protein of interest and of a small organic molecule, CrAsH. CrAsH becomes fluorescent if it binds to the tetracysteine tag. The part of the biosensor that enables detection by means of 129 Xe NMR spectroscopy, which is linked to the CrAsH moiety by a spacer, is based on a cryptophane core that is fully suited to reversibly host xenon. Three different peptides, containing the tetracysteine tag and four organic biosensors of different stereochemistry, are benchmarked to propose the best couple that is fully suited for the in vitro detection of proteins.

pH sensitivity of FRET reporters based on cyan and yellow fluorescent proteins.

It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by genetically encoded reporters suffer from strong pH sensitivities close to the physiological pH range. We studied the consequences of these pH responses on the intracellular signals of model Förster resonant energy transfer (FRET) tandems and FRET-based reporters of cAMP-dependent protein kinase activity (AKAR) expressed in the cytosol of living BHK cells, while changing the intracellular pH by means of the nigericin ionophore. Although the simultaneous pH sensitivities of the donor and the acceptor may mask each other in some cases, the magnitude of the perturbations can be very significant, as compared to the functional response of the AKAR biosensor. Replacing the CFP donor by the spectrally identical, but pH-insensitive Aquamarine variant (pK1/2 = 3.3) drastically modifies the biosensor pH response and gives access to the acid transition of the yellow acceptor. We developed a simple model of pH-dependent FRET and used it to describe the expected pH-induced changes in fluorescence lifetime and ratiometric signals. This model qualitatively accounts for most of the observations, but reveals a complex behavior of the cytosolic AKAR biosensor at acid pHs, associated to additional FRET contributions. This study underlines the major and complex impact of pH changes on the signal of FRET reporters in the living cell.

Development of an Efficient FRET-Based Ratiometric Uranium Biosensor.

The dispersion of uranium in the environment can pose a problem for the health of humans and other living organisms. It is therefore important to monitor the bioavailable and hence toxic fraction of uranium in the environment, but no efficient measurement methods exist for this. Our study aims to fill this gap by developing a genetically encoded FRET-based ratiometric uranium biosensor. This biosensor was constructed by grafting two fluorescent proteins to both ends of calmodulin, a protein that binds four calcium ions. By modifying the metal-binding sites and the fluorescent proteins, several versions of the biosensor were generated and characterized in vitro. The best combination results in a biosensor that is affine and selective for uranium compared to metals such as calcium or other environmental compounds (sodium, magnesium, chlorine). It has a good dynamic range and should be robust to environmental conditions. In addition, its detection limit is below the uranium limit concentration in drinking water defined by the World Health Organization. This genetically encoded biosensor is a promising tool to develop a uranium whole-cell biosensor. This would make it possible to monitor the bioavailable fraction of uranium in the environment, even in calcium-rich waters.

tdLanYFP, a Yellow, Bright, Photostable, and pH-Insensitive Fluorescent Protein for Live-Cell Imaging and Förster Resonance Energy Transfer-Based Sensing Strategies.

Yellow fluorescent proteins (YFPs) are widely used as optical reporters in Förster resonance energy transfer (FRET)-based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pH values. Here, we characterize the yellow fluorescent protein tdLanYFP, derived from the tetrameric protein from the cephalochordate Branchiostoma lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133,000 mol-1·L·cm-1, it is, to our knowledge, the brightest dimeric fluorescent protein available. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and in live cells. As a consequence, tdLanYFP allows imaging of cellular structures with subdiffraction resolution using STED nanoscopy and is compatible with the use of spectromicroscopies in single-molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pH values. Finally, we show that tdLanYFP is a valuable FRET partner either as a donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFP a very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging including FRET experiments at acidic pH.

Minimum set of mutations needed to optimize cyan fluorescent proteins for live cell imaging.

Cyan fluorescent proteins (CFPs) are widely used as FRET donors in genetically encoded biosensors for live cell imaging. Recently, cyan variants with greatly improved fluorescence quantum yields have been developed by large scale random mutagenesis. We show that the introduction of only two mutations, T65S and H148G, is able to confer equivalent performances on the popular form ECFP, leading to Aquamarine (QY = 0.89, τ(f) = 4.12 ns). Besides an impressive pH stability (pK(1/2) = 3.3), Aquamarine shows a very low general sensitivity to its environment, and undetectable photoswitching reactions. Aquamarine gives efficient and bright expression in different mammalian cell systems, with a long and single exponential intracellular fluorescence lifetime mostly insensitive to the fusion or the subcellular location of the protein. Aquamarine was also able to advantageously replace the CFP donor in the FRET biosensor AKAR for ratiometric measurements of protein kinase A activity. The performances of Aquamarine show that only two rounds of straightforward single point mutagenesis can be a quick and efficient way to optimize the donor properties in FRET-based biosensors.

The single T65S mutation generates brighter cyan fluorescent proteins with increased photostability and pH insensitivity.

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.