Stéphanie Finet

Polytech Paris-UPMC, Lutetia Parisorum, Île-de-France, France

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Publications (52)205.32 Total impact

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    ABSTRACT: The ubiquitous small heat shock proteins are essential elements in cellular protection, through a molecular chaperone activity. Among them, human small heat shock protein HspB1, HspB4 and HspB5 are involved in oncogenesis, anti-apoptotic activity and lens transparency. Therefore, these proteins are potential therapeutic targets in many diseases. Their general chaperone activity is related to their dynamic and multiple oligomeric structures, which are still poorly understood. The tissue selective distribution of HspB1 and HspB4, two cellular partners of HspB5, suggests that these two proteins might have evolved to play distinct physiological functions. Moreover, hetero-complex formation seems to be favoured in vivo, yet the functional specificity of the HspB1-HspB5 and HspB4-HspB5 hetero-complexes compared to the homo-oligomers remains unclear in the stress response pathway. A powerful approach combining biochemistry, biophysics and bioinformatics, allowed us to compare the different assemblies, with a special emphasis on the structural data, subunit exchange properties, activity and sequence evolution. We showed that they all exhibit different properties, from structural organization in physiological versus stress conditions, to chaperone-like activity, whatever the level of sequence conservation. Subunit exchange kinetics leading to HspB1-HspB5 or HspB4-HspB5 hetero-complex formation is also different between these two complexes: HspB5 exchanges more rapidly subunits with HspB1 than with HspB4. The relative sequence conservation in the sHSP superfamily does hide important structural heterogeneity and flexibility, which confer an enlarged range of different surface necessary to efficiently form complexes with various stress-induced cellular targets. Our data suggest that the formation of hetero-complexes could be an original evolutionary strategy to gain new cellular functions.
    Biochimie 12/2011; 94(4):975-84. DOI:10.1016/j.biochi.2011.12.018 · 3.12 Impact Factor
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    ABSTRACT: While molecular adaptation to high temperature has been extensively studied, the effect of hydrostatic pressure on protein structure and enzymatic activity is still poorly understood. We have studied the influence of pressure on both the quaternary structure and enzymatic activity of the dodecameric TET3 peptidase from Pyrococcus horikoshii. Small angle X-ray scattering (SAXS) revealed a high robustness of the oligomer under high pressure of up to 300 MPa at 25°C as well as at 90°C. The enzymatic activity of TET3 was enhanced by pressure up to 180 MPa. From the pressure behavior of the different rate-constants we have determined the volume changes associated with substrate binding and catalysis. Based on these results we propose that a change in the rate-limiting step occurs around 180 MPa.
    Archives of Biochemistry and Biophysics 09/2011; 517(2):104-10. DOI:10.1016/j.abb.2011.07.017 · 3.04 Impact Factor
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    ABSTRACT: Penicillin binding proteins (PBPs) catalyze essential steps in the biosynthesis of peptidoglycan, the main component of the bacterial cell wall. PBPs can harbor two catalytic domains, namely the glycosyltransferase (GT) and transpeptidase (TP) activities, the latter being the target for β-lactam antibiotics. Despite the availability of structural information regarding bi-functional PBPs, little is known regarding the interaction and flexibility between the TP and GT domains. Here, we describe the structural characterization in solution by small angle X-ray scattering (SAXS) of PBP1b, a bi-functional PBP from Streptococcus pneumoniae. The molecule is present in solution as an elongated monomer. Refinement of internal coordinates starting from a homology model yields models in which the two domains are in an extended conformation without any mutual contact compatible with the existence of restricted mobility.
    Biochemical and Biophysical Research Communications 02/2011; 405(1):107-11. DOI:10.1016/j.bbrc.2011.01.003 · 2.28 Impact Factor
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    ABSTRACT: t is well known that the formation of G-quadruplex by guanosine derivatives strongly depends on counter-ions. To investigate quadruplex stability in the presence of different counter-ions and in the absence of a covalent axial backbone, high-pressure X-ray diffraction experiments have been performed on lyotropic phases of guanosine 5’-monophosphate (GMP) in the form of sodium and ammonium salts. As a result, concentration-pressure phase diagrams were obtained in a pressure range from 1 bar to about 2 kbar, and the structural properties of the different phases derived. Interestingly, cholesteric (Ch) and hexagonal (H) columnar phases were found in both GMP salts, but during compression a reverse behavior was detected: in GMP sodium salt, pressure induces a H-Ch phase transition, while in GMP ammonium salt the induced transition is Ch-H. Different counter-ion stabilization, which control quadruplex length and the consequence of lateral interactions, probably explain such observation.
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    ABSTRACT: Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy. Enzymatic activity was also measured under pressure and after decompression. A global model, consistent with all measurements, discloses structural and functional details of the pressure-induced dissociation of the tetramer. Before dissociating, the pressurized protein adopts a conformational substate characterized by an expansion of its substrate binding pocket at the expense of a large neighboring hydrophobic cavity. This substate should be adopted by the enzyme during its catalytic mechanism, where the active site has to accommodate larger intermediates and product. The approach, combining several high-pressure techniques, offers a new (to our knowledge) means of exploring structural and functional properties of transient states relevant to protein mechanisms.
    Biophysical Journal 05/2010; 98(10):2365-73. DOI:10.1016/j.bpj.2010.01.058 · 3.83 Impact Factor
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    ABSTRACT: Aging of the lens is accompanied by extensive deamidation of the lens specific proteins, the crystallins. Deamidated crystallins are increased in the insoluble proteins and may contribute to cataracts. Deamidation has been shown in vitro to alter the structure and decrease the stability of human lens betaB1, betaB2 and betaA3-crystallin. Of particular interest, betaB2 mutants were constructed to mimic the effect of in vivo deamidations at the interacting interface between domains, at Q70 in the N terminal domain and at Q162, its C-terminal homologue. The double mutant was also constructed. We previously reported that deamidation at the critical interface sites decreased stability, while preserving the dimeric 3D structure. In the present study, dynamic light scattering, differential scanning calorimetry and small angle X-ray scattering were used to investigate the effect of deamidation on stability, thermal unfolding and aggregation. The bovine betaLb fraction was used for comparative analysis. The chaperone requirements of the various samples were determined using bovine alpha-crystallins as the chaperone. Deamidation at both interface Gln residues or at Q70, but not Q162, significantly lowered the temperature for unfolding and aggregation, which was rapidly followed by precipitation. This deamidation-induced aggregation and precipitation was not completely prevented by alpha-crystallin chaperone. A potential mechanism for cataract formation in vivo involving accumulation of deamidated beta-crystallin aggregates is discussed.
    Experimental Eye Research 02/2010; 90(6):688-98. DOI:10.1016/j.exer.2010.02.007 · 3.02 Impact Factor
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    ABSTRACT: We report on small-angle x-ray scattering measurements on liquid water aimed at characterizing the pressure evolution of its large-scale structure. Diffraction profiles have been fitted assuming a Lorentzian dependence on the exchanged momentum. As a result, we observe an anomalous behavior of the diffracted intensity that tends to disappear, increasing either the pressure or the temperature. This effect is discussed in detail and imputed to the ability of hydrostatic pressure to weaken hydrogen bonds.
    The Journal of Chemical Physics 11/2009; 131(19):194502. DOI:10.1063/1.3259882 · 3.12 Impact Factor
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    ABSTRACT: A long-range ordered organic/inorganic material is synthesized from a bis-silane, (EtO)(3)Si-(CH(2))(3)-NHCONH-C(6)H(4)-NHCONH-(CH(2))(3)-Si(OEt)(3). This crosslinked sol-gel solid exhibits a supramolecular organization via intermolecular hydrogen bonding interactions between urea groups (-NHCONH-) and covalent siloxane bonding, triple bond Si-O-Si triple bond. Time-resolved in situ X-ray measurements (coupling small- and wide-angle X-ray scattering techniques) are performed to follow the different steps involved in the synthetic process. A new mechanism based on the crystallization of the hydrolyzed species followed by their polycondensation in solid state is proposed.
    Small 02/2009; 5(4):503-10. DOI:10.1002/smll.200800254 · 7.51 Impact Factor
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    ABSTRACT: Mutation of the Arg120 residue in the human alphaB-crystallin sequence has been shown to be associated with a significant ability to aggregate in cultured cells and have an increased oligomeric size coupled to a partial loss of the chaperone-like activity in vitro. In the present study, static and dynamic light scattering, small-angle X-ray scattering, and size exclusion chromatography were used to follow the temperature and pressure induced structural transitions of human alphaB-crystallin and its R120G, R120D, and R120K mutants. The wild type alphaB-crystallin was known to progressively increase in size with increasing temperature, from 43 to 60 degrees C, before aggregating after 60 degrees C. The capacity to increase in size with temperature or pressure, while remaining soluble, had disappeared with the R120G mutant and was found to be reduced for the R120K and R120D mutants. The R120K mutant, which preserves the particle charge, was the less impaired. The deficit of quaternary structure plasticity was well correlated with the decrease in chaperone-like activity previously observed. However, the mutant ability to exchange subunits, measured with a novel anion exchange chromatography assay, was found to be increased, suggesting subtle relationships between structural dynamics and function. From molecular dynamic simulations, the R120 position appeared critical to conserve proper intra- and intersubunit interactions. In silico mutagenesis followed by simulated annealing of the known small heat shock protein 3D structures suggested a destabilization of the dimeric substructure by the R120 mutations. The whole of the results demonstrated the importance of the R120 residue for structural integrity, both static and dynamic, in relation with function.
    Biochemistry 02/2009; 48(2):442-53. DOI:10.1021/bi8014967 · 3.19 Impact Factor
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    Proceedings of the National Academy of Sciences 11/2008; 105(44). DOI:10.1073/pnas.0807839105 · 9.81 Impact Factor
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    ABSTRACT: Myelin is a multi-lamellar membrane surrounding neuronal axons and increasing their conduction velocity. When investigated by small-angle x-ray scattering (SAXS), the lamellar quasi-periodical arrangement of the myelin sheath gives rise to distinct peaks, which allow the determination of its molecular organization and the dimensions of its substructures. In this study we report on the myelin sheath structural determination carried out on a set of human brain tissue samples coming from surgical biopsies of two patients: a man around 60 and a woman nearly 90 years old. The samples were extracted either from white or grey cerebral matter and did not undergo any manipulation or chemical-physical treatment, which could possibly have altered their structure, except dipping them into a formalin solution for their conservation. Analysis of the scattered intensity from white matter of intact human cerebral tissue allowed the evaluation not only of the myelin sheath periodicity but also of its electronic charge density profile. In particular, the thicknesses of the cytoplasm and extracellular regions were established, as well as those of the hydrophilic polar heads and hydrophobic tails of the lipid bilayer. SAXS patterns were measured at several locations on each sample in order to establish the statistical variations of the structural parameters within a single sample and among different samples. This work demonstrates that a detailed structural analysis of the myelin sheath can also be carried out in randomly oriented samples of intact human white matter, which is of importance for studying the aetiology and evolution of the central nervous system pathologies inducing myelin degeneration.
    Physics in Medicine and Biology 10/2008; 53(20):5675-88. DOI:10.1088/0031-9155/53/20/007 · 2.92 Impact Factor
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    ABSTRACT: In colloidal systems, the interplay between the short range attraction and long-range repulsion can lead to a low density associated state consisting of clusters of individual particles. Recently, such an equilibrium cluster phase was also reported for concentrated solutions of lysozyme at low ionic strength and close to the physiological pH. Stradner et al. [(2004) Equilibrium cluster formation in concentrated protein solutions and colloids. Nature 432:492-495] found that the position of the low-angle interference peak in small-angle x-ray and neutron scattering (SAXS and SANS) patterns from lysozyme solutions was essentially independent of the protein concentration and attributed these unexpected results to the presence of equilibrium clusters. This work prompted a series of experimental and theoretical investigations, but also revealed some inconsistencies. We have repeated these experiments following the protein preparation protocols of Stradner et al. using several batches of lysozyme and exploring a broad range of concentrations, temperature and other conditions. Our measurements were done in multiple experimental sessions at three different high-resolution SAXS and SANS instruments. The low-ionic-strength lysozyme solutions displayed a clear shift in peak positions with concentration, incompatible with the presence of the cluster phase but consistent with the system of repulsively interacting individual lysozyme molecules. Within the decoupling approximation, the experimental data can be fitted using an effective interparticle interaction potential involving short-range attraction and long-range repulsion.
    Proceedings of the National Academy of Sciences 05/2008; 105(13):5075-80. DOI:10.1073/pnas.0711928105 · 9.81 Impact Factor
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    ABSTRACT: We present the first time-resolved investigation of motions of proteins in densely grafted layers of spherical polyelectrolyte brushes. Using small-angle x-ray scattering combined with rapid stopped-flow mixing, we followed the uptake of bovine serum albumin by poly(acrylic acid) layer with high spatial and temporal resolution. We find that the total amount of adsorbed protein scales with time as t(1/4). This subdiffusive behavior is explained on the basis of directed motion of the protein along the polyelectrolyte chains.
    Physical Review Letters 04/2008; 100(15):158301. DOI:10.1103/PhysRevLett.100.158301 · 7.51 Impact Factor
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    ABSTRACT: In the past two decades, the geometric pathways involved in the transformations between inverse bicontinuous cubic phases in amphiphilic systems have been extensively theoretically modeled. However, little experimental data exists on the cubic-cubic transformation in pure lipid systems. We have used pressure-jump time-resolved X-ray diffraction to investigate the transition between the gyroid QGII and double-diamond QDII phases in mixtures of 1-monoolein in 30 wt % water. We find for this system that the cubic-cubic transition occurs without any detectable intermediate structures. In addition, we have determined the kinetics of the transition, in both the forward and reverse directions, as a function of pressure-jump amplitude, temperature, and water content. A recently developed model allows (at least in principle) the calculation of the activation energy for lipid phase transitions from such data. The analysis is applicable only if kinetic reproducibility is achieved, at least within one sample, and achievement of such kinetic reproducibility is shown here, by carrying out prolonged pressure-cycling. The rate of transformation shows clear and consistent trends with pressure-jump amplitude, temperature, and water content, all of which are shown to be in agreement with the effect of the shift in the position of the cubic-cubic phase boundary following a change in the thermodynamic parameters.
    Langmuir 04/2008; 24(6):2331-40. DOI:10.1021/la7023378 · 4.38 Impact Factor
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    ABSTRACT: Poloxamers F88 (EO97PO39EO97) and P85 (EO27PO39EO27) are triblock copolymers of ethylene oxide (EO) and propylene oxide (PO), which have the same hydrophobic PO block. We studied aqueous solutions of these two copolymers by the conjoint use of differential scanning calorimetry (DSC), rheology, and small-angle X-ray scattering (SAXS). The results showed that the temperature-induced micellization of aqueous solutions of F88 and P85 was a progressive process followed by gelation for sufficiently concentrated samples. Gelation was due to the ordered packing of micelles under a hexagonal compact (HC) structure for P85 and a body-centered cubic (BCC) phase for F88. Importantly, the phase diagram of F88/P85 mixtures in water was elucidated and showed the destabilization of the HC phase upon addition of small amounts of F88.
    Langmuir 05/2007; 23(9):5085-92. DOI:10.1021/la062622p · 4.38 Impact Factor
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    ABSTRACT: Stopped-flow mixing coupled to small-angle X-ray scattering (SAXS) is an established technique for investigating structural kinetics in solution down to the millisecond range. More recently, the emphasis has shifted to the sub-millisecond range using continuous flow microfluidic mixing devices. The aim of this article is to review the present status and limitations when applying mixing techniques to a wide range of soft matter and biological systems. In the case of SAXS, special consideration of the mixing quality is necessary for a quantitative description of the scattered intensity. This is demonstrated through two representative examples involving protein refolding and micellar self-assembly.
    Advances in Colloid and Interface Science 12/2006; 127(1):9-18. DOI:10.1016/j.cis.2006.06.003 · 8.64 Impact Factor
  • Acta Crystallographica Section A Foundations of Crystallography 08/2006; 62. DOI:10.1107/S0108767306097273 · 2.07 Impact Factor
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    ABSTRACT: The direct lyotropic polymorphism of dodecyltrimethylammonium chloride (DTAC) was investigated by synchrotron X-ray diffraction at different water concentrations under compression up to 2 kbar, i.e., in the pressure intermediate range where interesting biophysical transformations occur and the functional characteristics of cell membranes are altered. The results show that pressure induces the transition from the hexagonal phase to the micellar Pm3n cubic phase in hydrated samples (c between 0.5 and 0.6, c being the weight concentration of lipid in the mixture) and the transition from the bicontinuous Ia3d cubic phase to the hexagonal phase in drier samples (c = 0.8). By increasing the pressure on very dry samples, a lamellar L(alpha) phase was observed to form transitorily at the Ia3d cubic-hexagonal phase transition. Phase compressibility and then the lipid and water partial molecular compressibilities were derived as a function of pressure and concentration. As a result, we assessed the very low compressibility of the hydration water within the lipid phases, and we demonstrated that the compressibility of DTAC is very dependent on pressure. Moreover, the molecular parameters of DTAC calculated in the different phases during compression confirmed that pressure induces small but continuous conformational changes, definitely different from the large changes observed in lipid molecules forming type II structures.
    The Journal of Physical Chemistry B 07/2006; 110(25):12410-8. DOI:10.1021/jp054467d · 3.38 Impact Factor
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    ABSTRACT: The liquid crystalline lamellar (L(alpha)) to double-diamond inverse bicontinuous cubic (Q(D)(pi)) phase transition for the amphiphile monoelaidin in excess water exhibits a remarkable sequence of structural transformations for pressure or temperature jumps. Our data imply that the transition dynamics depends on a coupling between changes in molecular shape and the geometrical and topological constraints of domain size. We propose a qualitative model for this coupling based on theories of membrane fusion via stalks and existing knowledge of the structure and energetics of bicontinuous cubic phases.
    Physical Review Letters 04/2006; 96(10):108102. DOI:10.1103/PhysRevLett.96.108102 · 7.51 Impact Factor

Publication Stats

2k Citations
205.32 Total Impact Points

Institutions

  • 2011
    • Polytech Paris-UPMC
      Lutetia Parisorum, Île-de-France, France
  • 1997–2011
    • French National Centre for Scientific Research
      • Centre de Biochimie Structurale
      Lutetia Parisorum, Île-de-France, France
  • 2010
    • Paris Diderot University
      Lutetia Parisorum, Île-de-France, France
  • 2009
    • Pierre and Marie Curie University - Paris 6
      Lutetia Parisorum, Île-de-France, France
  • 2003–2009
    • European Synchrotron Radiation Facility
      • Division of Experiments
      Grenoble, Rhône-Alpes, France
  • 2006
    • Università Politecnica delle Marche
      Ancona, The Marches, Italy
  • 2005
    • Aix-Marseille Université
      Marsiglia, Provence-Alpes-Côte d'Azur, France
  • 2004
    • Sincrotrone Trieste S.C.p.A.
      Trst, Friuli Venezia Giulia, Italy