Hélène Pasquier | Paris Sud XI University (original) (raw)

Papers by Hélène Pasquier

Lighting up life: fluorescent proteins and optogenetic sensors Fluorescent proteins of the green ... more Lighting up life: fluorescent proteins and optogenetic sensors Fluorescent proteins of the green fluorescent protein (GFP) family have given birth to a vast array of targetable optical sensors that play a major role in the deciphering of live cell chemistry. These genetically encoded reporters benefit from unequaled specificity and integration into the cellular machinery, while they gather withing less than 100 Å the three essential functions of a nanosensor, namely functionalization, chemical detection, and optical conversion. Their numerous applications and their performances directly stem from the photodynamics and photochemistry of the chromophore carried by GFPs. Owing to mutagenesis, spectroscopy and modeling tools, the way in which the GFP structural and conformationnal characteristics modulate their optical properties is better understood today. This mechanistic view will become ever more essential in the rational engineering of new probes for bioimaging.

ACS Sensors

Yellow fluorescent proteins (YFPs) are widely used as optical reporters in Förster resonance ener... more 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.

Methods and Applications in Fluorescence

New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to i... more New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to intensive engineering based on random mutagenesis and directed evolution. However, most of these newly developed FPs fail to achieve all the performances required for their bioimaging applications. The design of highly optimised FP-based reporters, simultaneously displaying appropriate colour, multimeric state, chromophore maturation, brightness, photostability and environmental sensitivity will require a better understanding of the structural and dynamic determinants of FP photophysics. The recent development of cyan fluorescent proteins (CFPs) like mCerulean3, mTurquoise2 and Aquamarine brings a different view on these questions, as in this particular case, a step by step evaluation of critical mutations has been performed within a family of spectrally identical and evolutionary close variants. These efforts have led to CFPs with quantum yields close to unity, near single exponential emission decays, high photostability and complete insensitivity to pH, making them ideal choices as energy transfer donors in FRET and FLIM imaging applications. During this process, it was found that a proper amino-acid choice at only two positions (148 and 65) is sufficient to transform the performances of CFPs: with the help of structural and theoretical investigations, we rationalise here how these two positions critically control the CFP photophysics, in the context of FPs derived from the Aequorea victoria species. Today, these results provide a useful toolbox for upgrading the different CFP donors carried by FRET biosensors. They also trace the route towards the de novo design of FP-based optogenetic devices that will be perfectly tailored to dedicated imaging and sensing applications.

Acta crystallographica. Section D, Structural biology, Dec 1, 2016

Until recently, genes coding for homologues of the autofluorescent protein GFP had only been iden... more Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent protein lanYFP from Branchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures of lanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV-visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chro...

Langmuir, 2015

Supporting information A) Proteomic data B) Spontaneous and catalyzed refolding kinetics C) Distr... more Supporting information A) Proteomic data B) Spontaneous and catalyzed refolding kinetics C) Distribution of K values in Langmuir-Freundlich isotherms D) Competition kinetics of dGFP between adsorption on NPs and refolding.

Analytical and bioanalytical chemistry, Jan 27, 2015

It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by gen... more 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 mod...

The Journal of Physical Chemistry B, 2005

The dynamics and electronic absorption spectrum of enhanced cyan fluorescent protein (ECFP), a mu... more The dynamics and electronic absorption spectrum of enhanced cyan fluorescent protein (ECFP), a mutant of green fluorescent protein (GFP), have been studied by means of a 1 ns molecular dynamics (MD) simulation. The two X-ray conformations A′ and B′ of ECFP were considered. The chromophore was assumed to be neutral, and all titratable residues were taken in their standard protonation state at neutral pH. The protein was embedded in a box of water molecules (and counterions). The first result is that the two conformations A′ and B′ are found to be stable all along the simulation. Then, an analysis of the hydrogen-bond networks shows strong differences between the two conformations in the surroundings of the nitrogen atom of the indolic part of the chromophore. This is partly due to the imperfection in the barrel near the His148 residue, which allows the access of one solvent molecule inside the protein in conformation A′. Finally, quantum mechanical calculations of the electronic transition energies of the chromophore in the charge cloud of the protein and solvent water molecules were performed using the TDDFT method on 160 snapshots extracted every 5 ps of the MD trajectories. It is found that conformations A′ and B′ exhibit very similar spectra despite different H-bond networks involving the chromophore. This similarity is related to the weak charge transfer involved in the electronic transition and the weak electrostatic field created by ECFP near the chromophore, within the hypotheses made in the present simulation.

The Journal of Physical Chemistry B, 2003

ABSTRACT Two different kinds of bisviologen-linked [Ru(bpy)3]2+ complexes bearing pyrrole groups ... more ABSTRACT Two different kinds of bisviologen-linked [Ru(bpy)3]2+ complexes bearing pyrrole groups were synthesized. We attempt to photopolymerize these dyad systems in the presence of an irreversible oxidative species (O2 or diazonium salts). The success of the photopolymerization appears to be strongly dependent on the design of the dyad, i.e., the relative arrangement among the [Ru(bpy)3]2+ chromophore, the viologen acceptor, and the polymerizable pyrrole groups. Electrochemical and photophysical characterization of the monomers and of the soluble photopolymer has been made. The intramolecular electron transfer from the 3MLCT excited state of the Ru chromophore to the viologen groups has been evidenced as well as the reverse process. We pay special attention to the photostability of the viologen moieties in the presence of O2. The photodegradation of the monoreduced viologen in the presence of O2 provides an emissive species at 546 nm. This photodegradation is not observed in the polymer system. Finally, it has been found that electroreductive precipitation of the photopolymer allows the coating of electrode surfaces with electroactive films exhibiting the redox features of the dyad.

The Journal of Physical Chemistry B, 2001

The photophysical properties of polypyridyl ruthenium(II) complexes substituted by pyrrole groups... more The photophysical properties of polypyridyl ruthenium(II) complexes substituted by pyrrole groups and of their corresponding soluble polymers synthesized either by electrooxidative or photoredox techniques are investigated. While their spectral properties and the temperature dependence of their lifetime are similar and close to that found for the parent [Ru(bpy) 3 ] 2+ complex, the emission quantum yields (Φ L) and the timeresolved emission decays are strongly affected by the polymer structure. Most of the polymers are characterized by a nonexponential emission decay that can be satisfactorily analyzed globally over different temperature in the framework of a distribution of decay rates. An important decrease in Φ L along with the increase of the width of the distribution of decay rates occurs as the degree of cross-linking is increased. The 3 MLCT excited states features are also substantially dependent on the length of the alkyl linkage between the pyrrole group and the Ru(II) complex as well as on the synthetic method of polymerization used. Furthermore, the accessibility of the excited states to quenchers as methyl-viologen (electron withdrawer) or N-methylphenothiazine (electron donor) is not significantly affected by the polymer structure indicating that the un-cross-linked polymers seem to adopt an extended coil-like structure. Using the photophysical data, attempts were made to obtain insights into the polymer architecture.

Synthetic Metals, 1996

The structure of mixed cationic/nonionic micelles of hexadecyltrimethylammonium chloride (CTAC1) ... more The structure of mixed cationic/nonionic micelles of hexadecyltrimethylammonium chloride (CTAC1) and Triton Xl00 (TX 100) was studied at the molecular level using both NMR and fluorescence measurements. In rich CTACt mixtures, the physical properties (intramicellar viscosity, polarity) of mixed micelles are similar to those of the CTAC1 micelles. The peculiar structure of TXt00 micelles proposed by Robson and Dennis (J. Phys. Chem., 81 (1977) 1075) seems to be broken for mole fractions higher than 0.33. In rich TX100 medium and at low CTAC1 content, an interaction between the quaternary ammonium group of CTAC1 and the phenyl ring of TX100 has been shown. In these mixtures, the mixed micellar structure are close to the TX100 one.

Proteins: Structure, Function, and Bioinformatics, 2010

Molecular dynamics (MD) and quantum mechanical calculations of the Cerulean green fluorescent pro... more Molecular dynamics (MD) and quantum mechanical calculations of the Cerulean green fluorescent protein (a variant of enhanced cyan fluorescent protein ECFP) at pH 5.0 and 8.0 are presented, addressing two questions arising from experimental results (Malo et al., Biochemistry 2007;46:9865-9873): the origin of the blue shift of absorption spectrum when the pH is decreased from 8.0 to 5.0, and the lateral chain orientation of the key residue Asp148. We demonstrate that the blue shift is reproduced assuming that a rotation around the single bond of the exocyclic ring of the chromophore takes place when the pH changes from 5.0 to 8.0. We find that Asp148 is protonated and inside the barrel at pH 5.0 in agreement with crystallographic data. However, the hydrogen bond pattern of Asp148 is different in simulations of the solvated protein and in the crystal structure. This difference is explained by a partial closing of the cleft between strands 6 and 7 in MD simulations. This study provides also a structure at pH 8.0: the Asp148 carboxylate group is exposed to the solvent and the chromophore is stabilized in the trans conformation by a tighter hydrogen bond network. This work gives some insight into the relationship between the pH and the chromophore conformation and suggests an interpretation of the very similar fluorescent properties of ECFP and ECFP/H148D. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Molecular BioSystems, 2013

Cyan fluorescent proteins (CFPs) are widely used as FRET donors in genetically encoded biosensors... more 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, t 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.

Langmuir, 1998

The excitation energy transfer from monomers to dimers of N, N'-dioctadecyl thiacyanine inco... more The excitation energy transfer from monomers to dimers of N, N'-dioctadecyl thiacyanine incorporated in Langmuir-Blodgett (LB) films of Cd-arachidate or dipalmitoyl phosphatidic acid was studied as a function of the dye concentration, ranging from 0.5 to 6 mol%, to ...

Inorganic Chemistry Communications, 2002

Two novel heteroditopic ligands in which the bidentate 2; 2 0-bipyridine (bpy) ligand is covalent... more Two novel heteroditopic ligands in which the bidentate 2; 2 0-bipyridine (bpy) ligand is covalently linked to one or two 2; 2 0 : 6 0 ; 2 00terpyridine (terpy) terdentate ligands have been prepared and characterized. The synthesis and the physico-chemical features of their corresponding complexes in which the bpy site is complexed by a ruthenium moiety are also reported.

Langmuir, 1998

The excitation energy transfer from monomers to dimers of N, N'-dioctadecyl thiacyanine inco... more The excitation energy transfer from monomers to dimers of N, N'-dioctadecyl thiacyanine incorporated in Langmuir-Blodgett (LB) films of Cd-arachidate or dipalmitoyl phosphatidic acid was studied as a function of the dye concentration, ranging from 0.5 to 6 mol%, to ...

Chemical Physics, 1996

The photophysicai properties of a recently synthesized molecule, the bis-9,9'-(2-ethyl-10-hexyl)a... more The photophysicai properties of a recently synthesized molecule, the bis-9,9'-(2-ethyl-10-hexyl)anthryl (BOA), have been studied with the parent molecule, the 9,9'-bianthryl (BA), in solvents of various polarity. The comparative analysis of the photophysical behaviour of these two probes has been achieved in order to emphasise environmental effects of the alkyl substituent on the aromatic chromophore. The results obtained in perfluoroalkane, reported for the first time in this paper, suggest that the charge transfer state (CT) of BA or BOA contributes to the fluorescence emission even in non polar solvents like cyclohexane. The fluorescence spectra of these two probes in perfluoroaikane have been introduced as standard spectra of the locally excited state form (LE) and used to obtain a new decomposition of the other overall fluorescence spectra into two emission components: one coming from the CT state and the second from the initial excited singlet state (LE). We have analysed the solute-solvent interactions not only in term of Stokes shift but also using the inhomogeneous band broadening parameter. The vibrational band decomposition found its validation in the agreement observed between the thermodynamic and spectroscopic properties of both excited states. A correlation between the inhomogeneous broadening and the Stokes shift of the charge transfer spectrum is exhibited. The existence of up to 50% of the CT state even in non polar solvents like cyclohexane has been shown. The dipole moment of the CT state of BA and of BOA can be estimated in the range of 6.2 to 10.3 D depending on the solvatochromic model used. It has been shown that the alkyl substituent of BOA destabilises the CT state and only four methylene groups of each lateral chain of BOA contribute to the local environment of the aromatic chromophore.

Lighting up life: fluorescent proteins and optogenetic sensors Fluorescent proteins of the green ... more Lighting up life: fluorescent proteins and optogenetic sensors Fluorescent proteins of the green fluorescent protein (GFP) family have given birth to a vast array of targetable optical sensors that play a major role in the deciphering of live cell chemistry. These genetically encoded reporters benefit from unequaled specificity and integration into the cellular machinery, while they gather withing less than 100 Å the three essential functions of a nanosensor, namely functionalization, chemical detection, and optical conversion. Their numerous applications and their performances directly stem from the photodynamics and photochemistry of the chromophore carried by GFPs. Owing to mutagenesis, spectroscopy and modeling tools, the way in which the GFP structural and conformationnal characteristics modulate their optical properties is better understood today. This mechanistic view will become ever more essential in the rational engineering of new probes for bioimaging.

ACS Sensors

Yellow fluorescent proteins (YFPs) are widely used as optical reporters in Förster resonance ener... more 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.

Methods and Applications in Fluorescence

New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to i... more New fluorescent proteins (FPs) are constantly discovered from natural sources, and submitted to intensive engineering based on random mutagenesis and directed evolution. However, most of these newly developed FPs fail to achieve all the performances required for their bioimaging applications. The design of highly optimised FP-based reporters, simultaneously displaying appropriate colour, multimeric state, chromophore maturation, brightness, photostability and environmental sensitivity will require a better understanding of the structural and dynamic determinants of FP photophysics. The recent development of cyan fluorescent proteins (CFPs) like mCerulean3, mTurquoise2 and Aquamarine brings a different view on these questions, as in this particular case, a step by step evaluation of critical mutations has been performed within a family of spectrally identical and evolutionary close variants. These efforts have led to CFPs with quantum yields close to unity, near single exponential emission decays, high photostability and complete insensitivity to pH, making them ideal choices as energy transfer donors in FRET and FLIM imaging applications. During this process, it was found that a proper amino-acid choice at only two positions (148 and 65) is sufficient to transform the performances of CFPs: with the help of structural and theoretical investigations, we rationalise here how these two positions critically control the CFP photophysics, in the context of FPs derived from the Aequorea victoria species. Today, these results provide a useful toolbox for upgrading the different CFP donors carried by FRET biosensors. They also trace the route towards the de novo design of FP-based optogenetic devices that will be perfectly tailored to dedicated imaging and sensing applications.

Acta crystallographica. Section D, Structural biology, Dec 1, 2016

Until recently, genes coding for homologues of the autofluorescent protein GFP had only been iden... more Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent protein lanYFP from Branchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures of lanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV-visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chro...

Langmuir, 2015

Supporting information A) Proteomic data B) Spontaneous and catalyzed refolding kinetics C) Distr... more Supporting information A) Proteomic data B) Spontaneous and catalyzed refolding kinetics C) Distribution of K values in Langmuir-Freundlich isotherms D) Competition kinetics of dGFP between adsorption on NPs and refolding.

Analytical and bioanalytical chemistry, Jan 27, 2015

It is generally acknowledged that the popular cyan and yellow fluorescent proteins carried by gen... more 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 mod...

The Journal of Physical Chemistry B, 2005

The dynamics and electronic absorption spectrum of enhanced cyan fluorescent protein (ECFP), a mu... more The dynamics and electronic absorption spectrum of enhanced cyan fluorescent protein (ECFP), a mutant of green fluorescent protein (GFP), have been studied by means of a 1 ns molecular dynamics (MD) simulation. The two X-ray conformations A′ and B′ of ECFP were considered. The chromophore was assumed to be neutral, and all titratable residues were taken in their standard protonation state at neutral pH. The protein was embedded in a box of water molecules (and counterions). The first result is that the two conformations A′ and B′ are found to be stable all along the simulation. Then, an analysis of the hydrogen-bond networks shows strong differences between the two conformations in the surroundings of the nitrogen atom of the indolic part of the chromophore. This is partly due to the imperfection in the barrel near the His148 residue, which allows the access of one solvent molecule inside the protein in conformation A′. Finally, quantum mechanical calculations of the electronic transition energies of the chromophore in the charge cloud of the protein and solvent water molecules were performed using the TDDFT method on 160 snapshots extracted every 5 ps of the MD trajectories. It is found that conformations A′ and B′ exhibit very similar spectra despite different H-bond networks involving the chromophore. This similarity is related to the weak charge transfer involved in the electronic transition and the weak electrostatic field created by ECFP near the chromophore, within the hypotheses made in the present simulation.

The Journal of Physical Chemistry B, 2003

ABSTRACT Two different kinds of bisviologen-linked [Ru(bpy)3]2+ complexes bearing pyrrole groups ... more ABSTRACT Two different kinds of bisviologen-linked [Ru(bpy)3]2+ complexes bearing pyrrole groups were synthesized. We attempt to photopolymerize these dyad systems in the presence of an irreversible oxidative species (O2 or diazonium salts). The success of the photopolymerization appears to be strongly dependent on the design of the dyad, i.e., the relative arrangement among the [Ru(bpy)3]2+ chromophore, the viologen acceptor, and the polymerizable pyrrole groups. Electrochemical and photophysical characterization of the monomers and of the soluble photopolymer has been made. The intramolecular electron transfer from the 3MLCT excited state of the Ru chromophore to the viologen groups has been evidenced as well as the reverse process. We pay special attention to the photostability of the viologen moieties in the presence of O2. The photodegradation of the monoreduced viologen in the presence of O2 provides an emissive species at 546 nm. This photodegradation is not observed in the polymer system. Finally, it has been found that electroreductive precipitation of the photopolymer allows the coating of electrode surfaces with electroactive films exhibiting the redox features of the dyad.

The Journal of Physical Chemistry B, 2001

The photophysical properties of polypyridyl ruthenium(II) complexes substituted by pyrrole groups... more The photophysical properties of polypyridyl ruthenium(II) complexes substituted by pyrrole groups and of their corresponding soluble polymers synthesized either by electrooxidative or photoredox techniques are investigated. While their spectral properties and the temperature dependence of their lifetime are similar and close to that found for the parent [Ru(bpy) 3 ] 2+ complex, the emission quantum yields (Φ L) and the timeresolved emission decays are strongly affected by the polymer structure. Most of the polymers are characterized by a nonexponential emission decay that can be satisfactorily analyzed globally over different temperature in the framework of a distribution of decay rates. An important decrease in Φ L along with the increase of the width of the distribution of decay rates occurs as the degree of cross-linking is increased. The 3 MLCT excited states features are also substantially dependent on the length of the alkyl linkage between the pyrrole group and the Ru(II) complex as well as on the synthetic method of polymerization used. Furthermore, the accessibility of the excited states to quenchers as methyl-viologen (electron withdrawer) or N-methylphenothiazine (electron donor) is not significantly affected by the polymer structure indicating that the un-cross-linked polymers seem to adopt an extended coil-like structure. Using the photophysical data, attempts were made to obtain insights into the polymer architecture.

Synthetic Metals, 1996

The structure of mixed cationic/nonionic micelles of hexadecyltrimethylammonium chloride (CTAC1) ... more The structure of mixed cationic/nonionic micelles of hexadecyltrimethylammonium chloride (CTAC1) and Triton Xl00 (TX 100) was studied at the molecular level using both NMR and fluorescence measurements. In rich CTACt mixtures, the physical properties (intramicellar viscosity, polarity) of mixed micelles are similar to those of the CTAC1 micelles. The peculiar structure of TXt00 micelles proposed by Robson and Dennis (J. Phys. Chem., 81 (1977) 1075) seems to be broken for mole fractions higher than 0.33. In rich TX100 medium and at low CTAC1 content, an interaction between the quaternary ammonium group of CTAC1 and the phenyl ring of TX100 has been shown. In these mixtures, the mixed micellar structure are close to the TX100 one.

Proteins: Structure, Function, and Bioinformatics, 2010

Molecular dynamics (MD) and quantum mechanical calculations of the Cerulean green fluorescent pro... more Molecular dynamics (MD) and quantum mechanical calculations of the Cerulean green fluorescent protein (a variant of enhanced cyan fluorescent protein ECFP) at pH 5.0 and 8.0 are presented, addressing two questions arising from experimental results (Malo et al., Biochemistry 2007;46:9865-9873): the origin of the blue shift of absorption spectrum when the pH is decreased from 8.0 to 5.0, and the lateral chain orientation of the key residue Asp148. We demonstrate that the blue shift is reproduced assuming that a rotation around the single bond of the exocyclic ring of the chromophore takes place when the pH changes from 5.0 to 8.0. We find that Asp148 is protonated and inside the barrel at pH 5.0 in agreement with crystallographic data. However, the hydrogen bond pattern of Asp148 is different in simulations of the solvated protein and in the crystal structure. This difference is explained by a partial closing of the cleft between strands 6 and 7 in MD simulations. This study provides also a structure at pH 8.0: the Asp148 carboxylate group is exposed to the solvent and the chromophore is stabilized in the trans conformation by a tighter hydrogen bond network. This work gives some insight into the relationship between the pH and the chromophore conformation and suggests an interpretation of the very similar fluorescent properties of ECFP and ECFP/H148D. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Molecular BioSystems, 2013

Cyan fluorescent proteins (CFPs) are widely used as FRET donors in genetically encoded biosensors... more 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, t 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.

Langmuir, 1998

The excitation energy transfer from monomers to dimers of N, N'-dioctadecyl thiacyanine inco... more The excitation energy transfer from monomers to dimers of N, N'-dioctadecyl thiacyanine incorporated in Langmuir-Blodgett (LB) films of Cd-arachidate or dipalmitoyl phosphatidic acid was studied as a function of the dye concentration, ranging from 0.5 to 6 mol%, to ...

Inorganic Chemistry Communications, 2002

Two novel heteroditopic ligands in which the bidentate 2; 2 0-bipyridine (bpy) ligand is covalent... more Two novel heteroditopic ligands in which the bidentate 2; 2 0-bipyridine (bpy) ligand is covalently linked to one or two 2; 2 0 : 6 0 ; 2 00terpyridine (terpy) terdentate ligands have been prepared and characterized. The synthesis and the physico-chemical features of their corresponding complexes in which the bpy site is complexed by a ruthenium moiety are also reported.

Langmuir, 1998

The excitation energy transfer from monomers to dimers of N, N'-dioctadecyl thiacyanine inco... more The excitation energy transfer from monomers to dimers of N, N'-dioctadecyl thiacyanine incorporated in Langmuir-Blodgett (LB) films of Cd-arachidate or dipalmitoyl phosphatidic acid was studied as a function of the dye concentration, ranging from 0.5 to 6 mol%, to ...

Chemical Physics, 1996

The photophysicai properties of a recently synthesized molecule, the bis-9,9'-(2-ethyl-10-hexyl)a... more The photophysicai properties of a recently synthesized molecule, the bis-9,9'-(2-ethyl-10-hexyl)anthryl (BOA), have been studied with the parent molecule, the 9,9'-bianthryl (BA), in solvents of various polarity. The comparative analysis of the photophysical behaviour of these two probes has been achieved in order to emphasise environmental effects of the alkyl substituent on the aromatic chromophore. The results obtained in perfluoroalkane, reported for the first time in this paper, suggest that the charge transfer state (CT) of BA or BOA contributes to the fluorescence emission even in non polar solvents like cyclohexane. The fluorescence spectra of these two probes in perfluoroaikane have been introduced as standard spectra of the locally excited state form (LE) and used to obtain a new decomposition of the other overall fluorescence spectra into two emission components: one coming from the CT state and the second from the initial excited singlet state (LE). We have analysed the solute-solvent interactions not only in term of Stokes shift but also using the inhomogeneous band broadening parameter. The vibrational band decomposition found its validation in the agreement observed between the thermodynamic and spectroscopic properties of both excited states. A correlation between the inhomogeneous broadening and the Stokes shift of the charge transfer spectrum is exhibited. The existence of up to 50% of the CT state even in non polar solvents like cyclohexane has been shown. The dipole moment of the CT state of BA and of BOA can be estimated in the range of 6.2 to 10.3 D depending on the solvatochromic model used. It has been shown that the alkyl substituent of BOA destabilises the CT state and only four methylene groups of each lateral chain of BOA contribute to the local environment of the aromatic chromophore.