Fabienne Merola

Allosteric transitions of Torpedo acetylcholine receptor in lipids, detergent and amphipols: molecular interactions vs. physical constraints

The binding of a fluorescent agonist to the acetycholine receptor from Torpedo electric organ has been studied by time‐resolved spectroscopy in three different environments: in native membrane fragments, in the detergent CHAPS, and after complexation by amphipathic polymers (‘amphipols’). Binding kinetics was similar in the membrane and in amphipols, demonstrating that the receptor can display unaltered allosteric transitions outside its natural lipid environment. In contrast, allosteric equilibria were strongly shifted towards the desensitized state in CHAPS. Therefore, the effect of CHAPS likely results from molecular interactions rather than from the loss of bulk physical properties of the membrane environment.

Complex fluorescence of the cyan fluorescent protein: comparisons with the H148D variant and consequences for quantitative cell imaging.

We have studied the fluorescence decays of the purified enhanced cyan fluorescent protein (ECFP, with chromophore sequence Thr-Trp-Gly) and of its variant carrying the single H148D mutation characteristic of the brighter form Cerulean. Both proteins exhibit highly complex fluorescence decays showing strong temperature and pH dependences. At neutral pH, the H148D mutation leads (i) to a general increase in all fluorescence lifetimes and (ii) to the disappearance of a subpopulation, estimated to be more than 25% of the total ECFP molecules, characterized by a quenched and red-shifted fluorescence. The fluorescence lifetime distributions of ECFP and its H148D mutant remain otherwise very similar, indicating a high degree of structural and dynamic similarity of the two proteins in their major form. From thermodynamic analysis, we conclude that the multiexponential decay of ECFP cannot be simply ascribed, as is generally admitted, to the slow conformational exchange characterized by NMR and X-ray crystallographic studies [Seifert, M. H., et al. (2002) J. Am. Chem. Soc. 124, 7932-7942; Bae, J. H., et al. (2003) J. Mol. Biol. 328, 1071-1081]. Parallel measurements in living cells show that these fluorescence properties in neutral solution are very similar to those of cytosolic ECFP.

Relationship between Homo-oligomerization of a Mammalian Olfactory Receptor and Its Activation State Demonstrated by Bioluminescence Resonance Energy Transfer

G-protein-coupled receptor homo-oligomerization has been increasingly reported. However, little is known regarding the relationship between activation of the receptor and its association/conformational states. The mammalian olfactory receptors (ORs) belong to the G protein-coupled receptor superfamily. In this study, the homo-oligomerization status of the human OR1740 receptor and its involvement in receptor activation upon odorant ligand binding were addressed by co-immunoprecipitation and bioluminescence resonance energy transfer approaches using crude membranes or membranes from different cellular compartments. For the first time, our data clearly show that mammalian ORs constitutively self-associate into homodimers at the plasma membrane level. This study also demonstrates that ligand binding mediates a conformational change and promotes an inactive state of the OR dimers at high ligand concentrations. These findings support and validate our previously proposed model of OR activation/inactivation based on the tripartite odorant-binding protein-odorant-OR partnership.

Excited State Dynamics of the Green Fluorescent Protein on the Nanosecond Time Scale

We have introduced a new algorithm in the parallel processing PMEMD module of the AMBER suite that allows MD simulations with a potential involving two coupled torsions. We have used this modified module to study the green fluorescent protein. A coupled torsional potential was adjusted on high accuracy quantum chemical calculations of the anionic chromophore in the first excited state, and several 15-ns-long MD simulations were performed. We have obtained an estimate of the fluorescence lifetime (2.2 ns) to be compared to the experimental value (3 ns), which is, to the best of our knowledge, the first theoretical estimate of that lifetime.

Cyan fluorescent protein carries a constitutive mutation that prevents its dimerization.

The tendency of GFP-like fluorescent proteins to dimerize in vitro is a permanent concern as it may lead to artifacts in FRET imaging applications. However, we have found recently that CFP and YFP (the couple of GFP variants mostly used in FRET studies) show no trace of association in the cytosol of living cells up to millimolar concentrations. In this study, we investigated the oligomerization properties of purified CFP, by fluorescence anisotropy and sedimentation velocity. Surprisingly, we found that CFP has a much weaker homoaffinity than other fluorescent proteins (K(d) ≥ 3 × 10(-3) M), and that this is due to the constitutive N146I mutation, originally introduced into CFP to improve its brightness.

Molecular Dynamics of Biological Probes by Fluorescence Correlation Microscopy with Two-Photon Excitation

We report on the application of fluorescence correlation microscopy under two-photon excitation of fluorophores of biological interest: FITC–dextran (MW, from 20 to 150 kDa), green fluorescent protein (MW, 27 kDa), and fluorescein (MW, 330 Da). Under these experimental conditions, the translational diffusion coefficients of these molecules in aqueous solutions derived from the fluorescence intensity autocorrelation function were determined for the first time and were found to be 24 × 10−7, 8.2 × 10−7, and 3 × 10−7 cm2 s−1 for 150-kDa FITC–dextran, green fluorescent protein, and fluorescein, respectively. These results are discussed in connection with previously reported results obtained by different methods. The great sensibility of the system has been applied to single-molecule detection of the smaller fluorophore, fluorescein.

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 (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.

Fluorescent Properties of Oligonucleotide-conjugated Thiazole Orange Probes¶

The fluorescence properties of thiazole orange, linked via a ( 1 ) hydrophobic alkyl or a ( 2 ) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca‐2′‐deoxyriboadenylate (dA15), are evaluated. Linkage at the phosphate group yields two stereoisomers, S‐isomer of the phosphorus chiral center (Sp) and R‐isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange–oligonucleotide conjugates (TO‐probes). Quantum yields of fluorescence (Φfl) of between 0.04 and 0.07 were determined for the single‐stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5′[CACT15CAC3′]); Φfl values of between 0.06–0.35 were determined for the double‐stranded conjugates. The Φfl value (0.17) of thiazole orange, 1‐(N,N′‐trimethylaminopropyl)‐4‐[3‐methyl‐2,3‐dihydro‐(benzo‐1,3‐thiazole)‐2‐methylidene]‐quinolinium iodide (TO‐Pro 1) in the presence of the oligonucleotide duplex (TO‐Pro 1: dA15·d5′[CACT15CAC3′] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady‐state and time‐resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time‐resolved studies on the four double‐stranded TO‐probes revealed that the fluorescent oligonucleotide–thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO‐probes in which the isomeric structure of the phosphate‐chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher Φfl values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest Φfl and the greatest fraction of the longest‐lived fluorescent thiazole orange species (in the hybrid form).

Are the Fluorescent Properties of the Cyan Fluorescent Protein Sensitive to Conditions of Oxidative Stress

The modifications induced by reactive oxygen species (ROS) on fluorescent proteins (FPs) may have important implications for live cell fluorescence imaging. Using quantitative γ‐radiolysis, we have studied the ROS‐induced biochemical and photophysical perturbations on recombinant cyan fluorescent protein (CFP). After oxidation by the ˙OH radical, the protein displays a modified RP‐HPLC elution profile, while the CFP fluorescence undergoes pronounced decreases in intensity and lifetime, without changes in its excitation and emission spectra. Meanwhile, the Förster resonant energy transfer (FRET) between the single W57 and the chromophore remains unperturbed. These results rule out a direct oxidation of the CFP chromophore and of W57 as well as major changes in the protein 3D structure, but show that new fluorescent forms associated to a higher level of dynamic quenching have been generated. Thus, strict in situ controls are required when CFP is to be used for FRET studies in situations of oxidative activity, or under strong illumination.

Fluorescence energy transfer in lipid vesicles. A time-resolved analysis using stretched exponentials

Abstract Fluorescence energy transfer in lipid vesicles between N-(7-nitrobenz-2-oxa-1,3,-diazol-4-yl-labelled phosphatidylethanolamine (acting as donor) and N-(lissamine-rhodamin B)-labelled phosphotidylethanolamine (acting as acceptor) was studied by steady state and time-resolved fluorescence quenching analysis. Both fluorescent phospholipids were incorporated as minor components in four different types of lipid vesicle: dipalmitoylphosphatidylglycerol vesicles in their Lβ gel phase at 20 °C and in their Lα liquid crystalline phase at 50 °C, and egg yolk phosphatidylethanolamine vesicles at 40 °C in their Lα liquid crystalline phase at pH 9.5 and in their HII inverted hexagonal phase at pH 5.0. The quenching of the donor fluorescence by energy transfer is diffusion controlled in all cases, except in the Lβ gel phase. The dimensionality and type of constraints imposed on diffusion are different in each case, with the most efficient diffusion-controlled quenching in the hexagonal phase.