Publications

2010

• Investigation of the mechanisms of regulations, activation, and deactivation of Guanylate Cyclase, the endogenous NO-receptor, and NO-sensors
  
  • Yoo Byung-Kuk
  , 2010. The endogenous NO receptor, soluble guanylate cyclase (sGC) is the subject of this thesis. This enzyme synthesizes cGMP after NO binding. The main tool used is time-resolved picosecond-nanosecond absorption spectroscopy. We have shown that the simultaneous binding of CO and activators (YC-1, Bay 41-2272) induce a 5c heme-CO, like NO, explaining the synergistic activation. We identified all steps of the sGC-NO interaction by measuring the dynamics from picosecond to second. This dynamics of the entire protein is compared with that of the isolated beta subunit (1-190) and bacterial NO sensors. A myoglobin mutant (H93C) was used as a model for the study of heme in the 4- and 5-coordinated states. Finally, we measured the band III absorption of Mb and Hb to measure the movement of the heme iron after NO binding. We also investigated a potential inhibitor and an endogenous ligand of sGC.
• Investigation of the Cell Membrane Architecture by Single-Molecule Tracking of Peptidic Toxins
  
  • Türkcan Silvan
  , 2010. The cellular membrane is a vital part of the cell, which plays a crucial role in many cellular processes, such as, signaling and trafficking, and pathologies. This thesis aims to investigate the architecture of the cell membrane. The study uses the motion of two membrane receptors that are exploited by bacterial toxins to probe the architecture. Advances in light microscopy techniques have shown that many membrane receptors do not diffuse freely in the membrane, but undergo confined or anomalous diffusion. Currently a few models compete to explain the confinement of the receptors, such as the Picket-Fence model, lipid rafts and protein aggregates. To investigate the membrane, lanthanide doped nanoparticles (Y0.6Eu0.4VO4) are coupled to two different peptidic pore-forming toxins, the α-toxin of C. septicum and the ǫ-toxin of C. perfingens. Single molecule tracking of receptor bound labeled toxins in the apical membrane of MDCK cells in a wide-field microscope reveals the receptor motion with sub-diffraction resolution of down to 10 nm. The α & ǫ-toxin receptors both undergo confined diffusion with similar diffusion coefficients of 0.16 ± 0.14 µm2/s in temporaly stable domains of 0.5 µm2. To analyze the receptor trajectories, we intro- duced a novel approach based on an inference method. Our only assumption is that the receptor moves according to the Langevin equation of motion. This method exploits the information of the ensemble of the trajectory and the quality of the extracted values is verified through simulations. Both receptors are confined in a spring-like potential with a spring constant of 0.45 pN/µm. Tracking after cholesterol depletion by cholesterol ox- idase and cytoskeleton depolymerization by Latrunculin B, shows that confinement of single receptors is cholesterol dependent and actin depolymerization does not influence the confinement. Using the nanoparticle labels as a hydrodynamic force amplifier in a liquid flow, tests the response of the receptor to an external force and indicates attach- ment of the confining domains to the cytoskeleton. Finally, a model for the confinement of the receptor is proposed, based on the hydrophobic coupling of the receptor and the surrounding bilayer which can explain the spring-like potential of the confining domain.
• Influence of absorption on the time of flight of the light going through a complex medium
  
  • Kervella M.
  • d'Abzac F.-X.
  • Hache François
  • Hespel L.
  • Dartigalongue Thibault
  , 2011, 89 (Supplement n° 1), pp.C1V89S1P048-C1V89S1P048-4. The aim of this work is to evaluate the infuence of absorption processes on the time of fight of light going through an absorbing and scattering thick medium (clouds, paints, gas cell, etc). In order to study statistical scattering and absorbing processes, we use a Monte-Carlo simulation code with temporal phase function and Debye modes. The main result is that absorption inside particles induces a decrease of the global time delay. © 2011 by the Author(s); licensee Accademia Peloritana dei Pericolanti, Messina, Italy. (10.1478/C1V89S1P048)
  DOI : 10.1478/C1V89S1P048
• The archaeal Xpf/Mus81/FANCM homolog Hef and the Holliday junction resolvase Hjc define alternative pathways that are essential for cell viability in Haloferax volcanii
  
  • Lestini Roxane
  • Allers Thorsten
  DNA Repair, Elsevier, 2010, 9 (9), pp.994-1002. (10.1016/j.dnarep.2010.06.012)
  DOI : 10.1016/j.dnarep.2010.06.012
• Multiphoton microscopy of engineered dermal substitutes: assessment of 3-D collagen matrix remodeling induced by fibroblast contraction.
  
  • Pena Ana-Maria
  • Fagot Dominique
  • Olive Christian
  • Michelet Jean-François
  • Galey Jean-Baptiste
  • Leroy Frédéric
  • Beaurepaire Emmanuel
  • Martin Jean-Louis
  • Colonna Anne
  • Schanne-Klein Marie-Claire
  Journal of Biomedical Optics, Society of Photo-optical Instrumentation Engineers, 2010, 15 (5). Dermal fibroblasts are responsible for the generation of mechanical forces within their surrounding extracellular matrix and can be potentially targeted by anti-aging ingredients. Investigation of the modulation of fibroblast contraction by these ingredients requires the implementation of three-dimensional in situ imaging methodologies. We use multiphoton microscopy to visualize unstained engineered dermal tissue by combining second-harmonic generation that reveals specifically fibrillar collagen and two-photon excited fluorescence from endogenous cellular chromophores. We study the fibroblast-induced reorganization of the collagen matrix and quantitatively evaluate the effect of Y-27632, a RhoA-kinase inhibitor, on dermal substitute contraction. We observe that collagen fibrils rearrange around fibroblasts with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA-kinase inhibitor. Moreover, we show that the inhibitory effects are reversible. Our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the extracellular matrix induced by fibroblast contraction or other processes. (10.1117/1.3503411)
  DOI : 10.1117/1.3503411
• Cell lineage reconstruction of early zebrafish embryos using label-free nonlinear microscopy
  
  • Olivier Nicolas
  • Luengo-Oroz Miguel Angel
  • Duloquin Louise
  • Faure Emmanuel
  • Savy Thierry
  • Veilleux Israël
  • Solinas Xavier
  • Débarre Delphine
  • Bourgine Paul
  • Santos Andrés
  • Peyriéras Nadine
  • Beaurepaire Emmanuel
  Science, American Association for the Advancement of Science (AAAS), 2010, 329 (5994), pp.967-71. Quantifying cell behaviors in animal early embryogenesis remains a challenging issue requiring in toto imaging and automated image analysis. We designed a framework for imaging and reconstructing unstained whole zebrafish embryos for their first 10 cell division cycles and report measurements along the cell lineage with micrometer spatial resolution and minute temporal accuracy. Point-scanning multiphoton excitation optimized to preferentially probe the innermost regions of the embryo provided intrinsic signals highlighting all mitotic spindles and cell boundaries. Automated image analysis revealed the phenomenology of cell proliferation. Blastomeres continuously drift out of synchrony. After the 32-cell stage, the cell cycle lengthens according to cell radial position, leading to apparent division waves. Progressive amplification of this process is the rule, contrasting with classical descriptions of abrupt changes in the system dynamics. (10.1126/science.1189428)
  DOI : 10.1126/science.1189428
• Contribution of Time-Resolved Absorption Spectroscopy to Study Biological Questions
  
  • Lamarre Isabelle
  • Martin Jean-Louis
  • Négrerie Michel
  • Yoo Byung-Kuk
  , 2011, pp.123. In this report, we illustrate through the study of two allosteric heme proteins the contribution of time-resolved absorption spectroscopy to the understanding of fundamental biological mechanisms. The first studied protein is the endogenous nitric oxide receptor (guanylate cyclase, sGC) whose activation and deactivation mechanisms are not yet fully resolved. We show that the rebinding of the proximal histidine occurs in similar to 100 picoseconds in sGC, which is the very first step of its deactivation following NO release. We also show that synergistic action of CO together with an allosteric activator induces the cleavage of the bond between heme iron and proximal histidine. The second one is the prototype of allosteric protein, the dioxygen transporter hemoglobin (Hb). In Hb, we show that the motion of the iron atom, central to the heme, moves in similar to 18 picoseconds after NO binding; this motion represents the very first step of the allosteric T -> R transition. (10.1007/978-3-642-17913-6_15)
  DOI : 10.1007/978-3-642-17913-6_15
• Procédé et dispositif d'analyse d'interactions moléculaires et utilisations
  
  • Türkcan Silvan
  • Allain Jean-Marc
  • Alexandrou Antigoni
  , 2010. L'invention se rapporte à un procédé d'analyse d'une interaction entre une première molécule et une deuxième molécule liée à une particule, comprenant les étapes suivantes : - mettre en contact la première molécule et la deuxième molécule liée à la particule dans des conditions rendant possible leur interaction, - appliquer un flux liquide déterminé sur la particule liée à la deuxième molécule, - observer un déplacement de la particule liée à la deuxième molécule sous l'action du flux appliqué, - analyser l'interaction en fonction du déplacement observé et du flux appliqué, la particule ayant une résistance hydrodynamique supérieure à celle de la première et/ou de la deuxième molécule, et un nombre de Péclet massique supérieur à 1 L'invention se rapporte également à un dispositif d'analyse d'une interaction entre une première molécule et au moins une deuxième molécule, ainsi qu'à l'utilisation du procédé ou du dispositif dans un criblage d'une molécule candidate pour le développement d'un médicament.
• RNA polymerase mutations that facilitate replication progression in the rep uvrD recF mutant lacking two accessory replicative helicases
  
  • Baharoglu Zeynep
  • Lestini Roxane
  • Duigou Stéphane
  • Michel Bénédicte
  Molecular Microbiology, Wiley, 2010, 77 (2), pp.324. We observed that cells lacking Rep and UvrD, two replication accessory helicases, and the recombination protein RecF are cryo-sensitive on rich medium. We isolated five mutations that suppress this LB-cryo-sensitivity and show that they map in the genes encoding the RNA polymerase subunits RpoB and RpoC. These rpoB (D444G, H447R and N518D) and rpoC mutants (H113R and P451L) were characterized. rpoBH447R and rpoBD444G prevent activation of the Prrn core promoter in rich medium, but only rpoBH447R also suppresses the auxotrophy of a relA spoT mutant (stringent-like phenotype). rpoCH113R suppresses the thermo-sensitivity of a greA greB mutant, suggesting that it destabilizes stalled elongation complexes. All mutations but rpoCP451L prevent R-loop formation. We propose that these rpo mutations allow replication in the absence of Rep and UvrD by destabilizing RNA Pol upon replication-transcription collisions. In a RecF+ context, they improve growth of rep uvrD cells only if DinG is present, supporting the hypothesis that Rep, UvrD and DinG facilitate progression of the replication fork across transcribed sequences. They rescue rep uvrD dinG recF cells, indicating that in a recF mutant replication forks arrested by unstable transcription complexes can restart without any of the three known replication accessory helicases Rep, UvrD and DinG. (10.1111/j.1365-2958.2010.07208.x)
  DOI : 10.1111/j.1365-2958.2010.07208.x
• Liquid lens approaches for simultaneous standard and extended depth of field imaging
  
  • Olivier Nicolas
  • Mozet W.T.
  • Mermillod-Blondin A.
  • Beaurepaire Emmanuel
  • Arnold C.B.
  , 2010, pp.CThT6. A tunable acoustic gradient lens is shown to provide depth-of-field switching at kilohertz rates in a nonlinear microscope. We demonstrate two modulation strategies; fast varifocus scanning during each pixel and pseudo-Bessel beam excitation. ©2009 Optical Society of America. (10.1364/CLEO.2010.CThT6)
  DOI : 10.1364/CLEO.2010.CThT6
• Antimicrobial compounds of 1,4- naphtoquinone structure
  
  • Myllykallio Hannu
  • Basta Tamara
  • Boum Yap
  • Liebl Ursula
  , 2010. The present invention relates to a compound having formula (I) wherein: R1 is chosen from the group consisting of: phenyl group, possibly substituted, -CH2-C6H4-R'1 group, R'1 being chosen from the group consisting of: H, -OH, halogen, alkyl, aryl, CHO, -CN, -NO2, -SRalpha, -ORalpha, -NRalphaRbeta, -CONRalphaRbeta, -COORalpha, and -NHCORalpha, Ralpha and Rbeta representing independently from each other H, an alkyl group or an aryl group, R'1 being preferably in para position, and -CH2-CO-R' group, R' representing an aryl or heteroaryl group, said aryl and heteroaryl groups being possibly substituted, R2 is chosen from the group consisting of: -OH and halogen, and R3, R4, R5 and R6 are in particular H, for its use for the prevention and/or the treatment of bacterial infections
• Sickling of red blood cells in microfluidic droplets
  
  • Abbyad Paul
  • Baroud Charles N.
  • Martin Jean-Louis
  • Tharaux Pierre-Louis
  • Alexandrou Antigoni
  , 2010, 239. Red blood cell sickling associated with sickle cell disease is affected by a complex interplay of vascular components and conditions which are not yet fully understood. A primary trigger of sickling is low oxygen partial pressure. Therefore, we have developed a microfluidic device to rapidly vary the oxygen partial pressure within flowing microdroplets. By using a carrier oil at a known oxygen partial pressure, a microdroplet was subjected to a deoxygenation at a rate that depends on both flow conditions and droplet size. The oxygen concentration in a single droplet was characterized with a ruthenium compound whose fluorescence lifetime is proportional to the oxygen concentration. The device was shown to provoke sickling of individual red blood cells which was readily detected by birefringence. The versatility of the same device was also demonstrated by initiating a chemical reaction in a microdroplet using a gaseous reactant.
• Laser solide à 465 nm pour l'excitation de nano-particules d'Eu:YVO4
  
  • Castaing Marc
  • Balembois François
  • Georges Patrick
  • Türkcan Silvan
  • Nguyên Thanh-Liêm
  • Alexandrou Antigoni
  • Mialon G.
  • Gacoin Thierry
  • Boilot Jean-Pierre
  , 2010.
• Harmonic microscopy of isotropic and anisotropic microstructure of the human cornea
  
  • Olivier Nicolas
  • Aptel Florent
  • Plamann Karsten
  • Schanne-Klein Marie-Claire
  • Beaurepaire Emmanuel
  Optics Express, Optical Society of America - OSA Publishing, 2010, 18 (5), pp.5028-5040. In this study we present combined third-harmonic generation (THG) and second-harmonic generation (SHG) microscopy images of intact human corneas, and we analyze experimentally and theoretically the origin of the THG signal. Multiharmonic microscopy provides detailed images of the cornea microstructure over its entire thickness. A component of the THG signal originates from cellular structures and another one originates from anisotropy changes between successive collagen lamellae in the stroma. This anisotropy-related signal can be specifically detected using circular incident polarization, and provide contrasted images of the stacking and tissue-scale heterogeneity of stromal lamellae. Forward-radiated THG and SHG signals are generally anticorrelated, indicating that maximum THG is obtained from lamellar interfaces whereas maximum SHG is obtained from within lamellae. Polarization-resolved THG imaging reflects the a ernate anisotropy directions of the lamellae. We present a model for THG imaging of layered anisotropic samples and numerical calculations that account for our observations. (10.1364/OE.18.005028)
  DOI : 10.1364/OE.18.005028
• Method and device for acquiring signals in laser scanning microscopy (extension internationale du brevet EP20100708273)
  
  • Beaurepaire Emmanuel
  • Veilleux Israel
  • Olivier Nicolas
  • Debarre Delphine
  • Martin Jean-Louis
  , 2010. A method for acquiring signals in laser scanning microscopy, includes the steps of: moving a focused optical excitation beam relative to an object to be measured so that the focus point of the beam follows a predetermined path in the space of the object; and acquiring optical measurement signals along the path according to at least one acquisition parameter; characterised in that the path of the excitation beam is determined so as to substantially minimise the variations of the optical properties of at least one portion of the environments crossed by the excitation beam between consecutive acquisitions, and in that at least one acquisition parameter among the acquisition parameters is modulated during the movement of the excitation beam. A device for implementing the method is also described.
• Multiphoton microscopy of engineered dermal substitutes: Assessment of 3D collagen matrix remodeling induced by fibroblasts contraction
  
  • Pena A.-M.
  • Olive C.
  • Michelet J.-F.
  • Galey J.-B.
  • Fagot D.
  • Leroy F.
  • Martin Jean-Louis
  • Colonna A.
  • Schanne-Klein Marie-Claire
  , 2010, 7548, pp.1. One of the main functions of dermal fibroblasts is the generation of mechanical forces within their surrounding extracellular matrix. Investigating molecules that could modulate fibroblast contraction and act as potent anti aging ingredients requires the development of three-dimensional in situ imaging methodologies for dermal substitute analysis. Here we use multiphoton microscopy in order to investigate the fibroblast-induced collagen matrix reorganization in engineered dermal tissue and to evaluate the effect of Y27632, a RhoA kinase inhibitor on dermal substitutes contraction. We observe that collagen fibrils rearrange around fibroblast with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA kinase inhibitor. Moreover, when the culture medium containing the inhibitor was replaced with a control medium, the dermal substitutes presented the same 3D reorganization as the control samples, which indicates that the inhibitory effects are reversible. In conclusion, our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the matrix induced by fibroblast contraction. © 2010 Copyright SPIE - The International Society for Optical Engineering. (10.1117/12.839947)
  DOI : 10.1117/12.839947
• Nonlinear optical response of the collagen triple helix and second harmonic microscopy of collagen liquid crystals
  
  • Deniset-Besseau Ariane
  • de Sa Peixoto Paolo
  • Duboisset J.
  • Loison Claire
  • Hache François
  • Benichou E.
  • Brevet P.-F.
  • Mosser G.
  • Schanne-Klein Marie-Claire
  , 2010, 7569, pp.Second Harmonic Generation II. Collagen is characterized by triple helical domains and plays a central role in the formation of fibrillar and microfibrillar networks, basement membranes, as well as other structures of the connective tissue. Remarkably, fibrillar collagen exhibits efficient Second Harmonic Generation (SHG) and SHG microscopy proved to be a sensitive tool to score fibrotic pathologies. However, the nonlinear optical response of fibrillar collagen is not fully characterized yet and quantitative data are required to further process SHG images. We therefore performed Hyper-Rayleigh Scattering (HRS) experiments and measured a second order hyperpolarisability of 1.25 10-27 esu for rat-tail type I collagen. This value is surprisingly large considering that collagen presents no strong harmonophore in its amino-acid sequence. In order to get insight into the physical origin of this nonlinear process, we performed HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-Gly)10]3. It showed that the collagen large nonlinear response originates in the tight alignment of a large number of weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal along the triple helix. To illustrate this mechanism, we successfully recorded SHG images in collagen liquid solutions by achieving liquid crystalline ordering of the collagen triple helices. © 2010 Copyright SPIE - The International Society for Optical Engineering. (10.1117/12.840873)
  DOI : 10.1117/12.840873
• Nonlinear optical imaging of lyotropic cholesteric liquid crystals.
  
  • Deniset-Besseau Ariane
  • de Sa Peixoto Paolo
  • Mosser G.
  • Schanne-Klein Marie-Claire
  Optics Express, Optical Society of America - OSA Publishing, 2010, 18 (2), pp.1113-1121. We use nonlinear optical microscopy combining Second Harmonic Generation (SHG) microscopy and Two-Photon Excited Fluorescence (2PEF) signals to characterize collagen lyotropic liquid crystals. We show that SHG signals provide highly contrasted images of the three-dimensional texture of cholesteric patterns with submicrometer lateral resolution. Moreover, simultaneous recording of the 2PEF signal enables in situ quantitative mapping of the molecular concentration and its correlation with the observed textures. We apply this technique to the characterization of biomimetic textures obtained in concentrated collagen liquid solutions. We successfully image biologically relevant organizations that are similar to the collagen organization found as a stabilized state in compact bones. (10.1364/OE.18.001113)
  DOI : 10.1364/OE.18.001113
• Multimodal Nonlinear Imaging of the Human Cornea
  
  • Aptel Florent
  • Olivier Nicolas
  • Deniset-Besseau Ariane
  • Legeais Jean-Marc
  • Plamann Karsten
  • Schanne-Klein Marie-Claire
  • Beaurepaire Emmanuel
  Investigative Ophthalmology & Visual Science, Association for Research in Vision and Ophthalmology, 2010, 51 (5), pp.2459-2465. Purpose: to evaluate the potential of third-harmonic generation (THG) microscopy combined with second-harmonic generation (SHG) and two-photon excited fluorescence (2PEF) microscopies for visualizing the microstructure of the human cornea and trabecular meshwork based on their intrinsic nonlinear properties. Methods: fresh human corneal buttons and corneoscleral discs from an eye bank were observed under a multiphoton microscope incorporating a titanium-sapphire laser and an optical parametric oscillator for the excitation, and equipped with detection channels in the forward and backward directions. Results: original contrast mechanisms of THG signals in cornea with physiological relevance were elucidated. THG microscopy with circular incident polarization detected microscopic anisotropy and revealed the stacking and distribution of stromal collagen lamellae. THG imaging with linear incident polarization also revealed cellular and anchoring structures with micrometer resolution. In edematous tissue, a strong THG signal around cells indicated the local presence of water. Additionally, SHG signals reflected the distribution of fibrillar collagen, and 2PEF imaging revealed the elastic component of the trabecular meshwork and the fluorescence of metabolically active cells. Conclusions: the combined imaging modalities of THG, SHG, and 2PEF provide key information about the physiological state and microstructure of the anterior segment over its entire thickness with remarkable contrast and specificity. This imaging method should prove particularly useful for assessing glaucoma and corneal physiopathologies. (10.1167/iovs.09-4586)
  DOI : 10.1167/iovs.09-4586
• Heme-heme and heme-ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy
  
  • Rappaport Fabrice
  • Zhang Jie
  • Vos Marten H.
  • Gennis Robert B.
  • Borisov Vitaliy B.
  Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier, 2010, 1797 (9), pp.1657-1664. Cytochrome bd is a terminal quinol:O(2) oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b(558), b(595), and d. The role of heme b(595) remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d-CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b(595) causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b(595) and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with tau similar to 12 mu s, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with tau similar to 14 ns, 14 mu s, and 280 mu s. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-mu s component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in similar to 4% of the enzyme population. The final, 280-mu s component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b(595), and not that of heme b(558), controls the pathway(s) by which CO migrates between heme d and the medium. Cop 2010 Elsevier B.V. All rights reserved. (10.1016/j.bbabio.2010.05.010)
  DOI : 10.1016/j.bbabio.2010.05.010
• D1 protein variants in Photosystem II from Thermosynechococcus elongatus studied by low temperature optical spectroscopy
  
  • Hughes Joseph L.
  • Cox Nicholas
  • Rutherford A.William
  • Krausz Elmars
  • Lai Thanh-Lan
  • Boussac Alain
  • Sugiura Miwa
  Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics, Elsevier, 2010, 1797 (1), pp.11-19. In Photosystem II (PSII) from Thermosynechococcus elongatus, high-light intensity growth conditions induce the preferential expression of the psbA3 gene over the psbA1 gene. These genes encode for the D1 protein variants labeled D1:3 and D1:1, respectively. We have compared steady state absorption and photo-induced difference spectra at < 10 K of PSII containing either D1:1 or D1:3. The following differences were observed. (i) The pheophytin Qx band was red-shifted in D1:3 (547.3 nm) compared to D1:1 (544.3 nm). (ii) The electrochromism on the PheoD1 Qx band induced by QA− (the C550 shift) was more asymmetric in D1:3. (iii) The two variants differed in their responses to excitation with far red (704 nm) light. When green light was used there was little difference between the two variants. With far red light the stable (t1/2 > 50 ms) QA− yield was ∼ 95% in D1:3, and ∼ 60% in D1:1, relative to green light excitation. (iv) For the D1:1 variant, the quantum efficiency of photo-induced oxidation of side-pathway donors was lower. These effects can be correlated with amino acid changes between the two D1 variants. The effects on the pheophytin Qx band can be attributed to the hydrogen bond from Glu130 in D1:3 to the 131-keto of PheoD1, which is absent for Gln130 in D1:1. The reduced yield with red light in the D1:1 variant could be associated with either the Glu130Gln change, and/or the four changes near the binding site of PD1, in particular Ser153Ala. Photo-induced QA− formation with far red light is assigned to the direct optical excitation of a weakly absorbing charge transfer state of the reaction centre. We suggest that this state is blue-shifted in the D1:1 variant. A reduced efficiency for the oxidation of side-pathway donors in the D1:1 variant could be explained by a variation in the location and/or redox potential of P+. (10.1016/j.bbabio.2009.07.007)
  DOI : 10.1016/j.bbabio.2009.07.007
• Picosecond primary structural transition of the heme is retarded after nitric oxide binding to heme proteins
  
  • Kruglik Sergei G.
  • Yoo Byung-Kuk
  • Franzen S.
  • Vos Marten H.
  • Martin Jean-Louis
  • Négrerie Michel
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2010, 107 (31), pp.13678. We investigated the ultrafast structural transitions of the heme induced by nitric oxide (NO) binding for several heme proteins by subpicosecond time-resolved resonance Raman and femtosecond transient absorption spectroscopy. We probed the heme iron motion by the evolution of the iron-histidine Raman band intensity after NO photolysis. Unexpectedly, we found that the heme response and iron motion do not follow the kinetics of NO rebinding. Whereas NO dissociation induces quasi-instantaneous iron motion and heme doming (< 0.6 ps), the reverse process results in a much slower picosecond movement of the iron toward the planar heme configuration after NO binding. The time constant for this primary domed-to-planar heme transition varies among proteins (∼30 ps for myoglobin and its H64V mutant, ∼15 ps for hemoglobin, ∼7 ps for dehaloperoxidase, and ∼6 ps for cytochrome c) and depends upon constraints exerted by the protein structure on the heme cofactor. This observed phenomenon constitutes the primary structural transition in heme proteins induced by NO binding. (10.1073/pnas.0912938107)
  DOI : 10.1073/pnas.0912938107
• Expression and purification of untagged and histidine-tagged folate-dependent tRNA:m5U54 methyltransferase from Bacillus subtilis
  
  • Hamdane Djemel
  • Skouloubris Stéphane
  • Myllykallio Hannu
  • Golinelli-Pimpaneau Béatrice
  Protein Expression and Purification, Elsevier, 2010, 73 (1), pp.83-89. Folate-dependent tRNA m5U methyltransferase TrmFO is a flavoprotein that catalyzes the C5-methylation of uridine at position 54 in the T-Psi-C loop of tRNA in several bacteria. Here we report the cloning and optimization of expression in Escherichia coli BL21 (DE3) of untagged, N-terminus, C-terminus (His)6-tagged TrmFO from Bacillus subtilis. Tagged and untagged TrmFO were purified to homogeneity by metal affinity or ion exchange and heparin affinity, respectively, followed by size-exclusion chromatography. The tag did not significantly alter the expression level, flavin content, activity and secondary structure of the protein. Cop. 2010 Elsevier Inc. All rights reserved. (10.1016/j.pep.2010.04.013)
  DOI : 10.1016/j.pep.2010.04.013
• Imagerie de la cornée et de découpes laser par tomographie optique cohérente (OCT) et microscopie multiphoton
  
  • Latour Gael
  • Georges Gaelle
  • Siozade Laure
  • Deumié Carole
  • Schanne-Klein Marie-Claire
  , 2010.
• Quantifying Biomolecule Diffusivity Using an Optimal Bayesian Method
  
  • Voisinne Guillaume
  • Alexandrou Antigoni
  • Masson Jean-Baptiste
  Biophysical Journal, Biophysical Society, 2010, 98 (4), pp.596-605. We propose a Bayesian method to extract the diffusivity of biomolecules evolving freely or inside membrane microdomains This approach assumes a model of motion for the paticle considered, namely free Brownian motion or confined diffusion In each framework, a systematic Bayesian scheme is provided for estimating the diffusivity We show that this method reaches the best performances theoretically achievable Its efficiency overcomes that of widely used methods based on the analysis of the mean-square displacement The approach presented here also gives direct access to the uncertainty on the estimation of the diffusivity and predicts the number of steps of the trajectory necessary to achieve any desired precision Its robustness with respect to noise on the position of the biomolecule is also investigated (10.1016/j.bpj.2009.10.051)
  DOI : 10.1016/j.bpj.2009.10.051