Laboratoire d'optique et biosciences
Laboratoire d'optique et biosciences
Laboratoire d'optique et biosciences

Articles

  • Processus ultra-rapides dans les hémoprotéines [Ultrafast processes in heme proteins]
    • Vos Marten H.
    L'Actualité Chimique, Société chimique de France (SCF) , 2008, 317 (52), pp.55 . Les hémoprotéines sont impliquées dans une grande diversité de fonctions biologiques qui incluent transport et stockage d'oxygène, catalyse, transfert d'électron et signalisation. Le fer de l'hème peut être lié aux acides aminés et également aux molécules diatomiques (O2, NO, CO). Ces liaisons peuvent être rompues par une impulsion lumineuse avec un rendement élevé. En utilisant des techniques de spectroscopie optique ultrarapide, cette propriété donne la possibilité unique d'étudier la dynamique structurelle et électronique de l'hème et de son environnement protéique à l'échelle femtoseconde-picoseconde, c'est-à-dire à l'échelle même des vibrations internes de la macromolécule. Cet article décrit des développements récents dans ce domaine, en particulier l'implication de mouvements concertés hème-protéine dans des réactions balistiques de transfert de ligands et la possibilité de suivre des étapes intermédiaires de propagation d'un " signal " au sein de protéines de signalisation. Heme proteins contribute to a large variety of biological functions including transport and storage of oxygen, catalysis, electron transfer and signalling. The iron atom in the heme can bind amino acids and also diatomic molecules (O-2, NO, CO). These bonds can efficiently be broken by a light pulse. Using ultrafast optical spectroscopy techniques, this property allows the study of structural and electronic dynamics in the heme and its proteic environment at femtosecond-picosecond time scales, i.e. at the time scale of internal vibrations of the macromolecular systems. This review describes recent progress in this domain, specifically the role of concerted movements of the heme-protein system in ballistic ligand transfer reactions as well as the real-time monitoring of signal propagation within signalling proteins.
  • Fast ligand and electron transfer dynamics in oxidases and cytochrome c
    • Vos Marten H.
    Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics, Elsevier , 2008, 1777 (Supplement), pp.S66 . The active site of heme-copper oxidases contains two cofactors, heme a3 and CuB, which can both bind external ligands as the substrate O2 and signalling molecules NO and CO, and which are both involved in electron transfer processes. Over the last few years we have exploited the fact that the heme-ligand bond can be dissociated by a short light pulse to explore the dynamics of CO and NO in the active site and the interaction between the two cofactors using ultrafast spectroscopic techniques. For example, we have time-resolved the CO transfer from heme a3 to CuB and shown that it occurs in a ballistic way in ∼ 500 fs, which presumably reflects rigidity of the active site. Heme a is located close to heme a3 (∼ 7 Å edge-to-edge) and acts as electron donor for the active site. Using mixed valence oxidases we have extended the 'reverse electron flow' technique to the ultrafast regime and demonstrated that this electron transfer process occurs in only 1.2 ns. The process is activationless and associated with a very low reorganization energy (< 200 meV), in contrast to common assumptions but in general agreement with the hydrophobic environment of the reactants. Finally, ligand dynamics in native and modified cytochrome c reflects the rigidity required for optimal electron transfer properties. (10.1016/j.bbabio.2008.05.259)
    DOI : 10.1016/j.bbabio.2008.05.259
  • Two photon-induced electron injection from a nanotrigger in native endothelial NO-synthase
    • Beaumont Edward
    • Lambry Jean-Christophe
    • Robin A.-C.
    • Martasek P.
    • Blanchard-Desce M.
    • Slama-Schwok Anny
    ChemPhysChem, Wiley-VCH Verlag , 2008, 9 (16), pp.2325 . We have recently designed a nanotrigger (NT), a photoactive molecule addressing the NADPH sites of proteins. This nanotrigger has a 103 times larger two-photon cross-section compared to the ubiquitous NADPH cofactor. In this work, we tested whether two-photon excitation of the bound NT to NADPH sites may be used to initiate enzymatic catalysis by appropriate electron injection. To establish proof of principle, we monitored the ultrafast absorption of NT bound to the fully active endothelial NO-Synthase (eNOS) following excitation by one and two-photons at 405 and 810 nm, respectively. Electron injection from NT* to FAD in eNOS initiated the catalytic cycle in 15±3 ps at both exciting wavelengths. The data proved for the first time that electron transfer can be promoted by two-photon excitation. We also show that the nanotrigger decays faster in homogeneous solvents than in the NADPH site of proteins, suggesting that hindered environments modified the natural decay of NT. The nanotrigger provides a convenient way of synchronizing an ensemble of proteins in solution with a femtosecond laser pulse. The ability of NT to initiate NOS catalysis by two-photon excitation may be exploited for controlled and localized release of free NO in cells with enhanced spatial and temporal resolution. Cop. 2008 Wiley-VCH Verlag GmbH & Co. KGaA. (10.1002/cphc.200800411)
    DOI : 10.1002/cphc.200800411
  • Mechanical factors activate beta-catenin-dependent oncogene expression in APC1638N/+ mouse colon
    • Whitehead J.
    • Vignjevic Danijela
    • Fütterer C.
    • Beaurepaire Emmanuel
    • Robine Sylvie
    • Farge Emmanuel
    HFSP Journal , 2008, 2 (5), pp.286 . catenin acts as a critical regulator of gastrointestinal homeostasis through its control of the Wnt signaling pathway, and genetic or epigenetic lesions which activate Wnt signaling are the primary feature of colon cancer. -catenin is also a key element of mechanotranscription pathways, leading to upregulation of master developmental gene expression during Drosophila gastrulation, or regulating mammalian bone development and maintenance. Here we investigate the impact of mechanical stimulation on the initiation of colon cancer. Myc and Twist1, two oncogenes regulated through -catenin, are expressed in response to transient compression in APC deficient "APC1638N/+... colon tissue explants, but not in wild-type colon explants. Mechanical stimulation of APC1638N/+ tissue leads to the phosphorylation of -catenin at tyrosine 654, the site of interaction with E-cadherin, as well as to increased nuclear localization of -catenin. The mechanical activation of Myc and Twist1 expression in APC1638N/+ colon can be prevented by blocking -catenin phosphorylation using Src kinase inhibitors. Microenvironmental signals are known to cooperate with genetic lesions to promote the nuclear -catenin accumulation which drives colon cancer. Here we demonstrate that when APC is limiting, mechanical strain, such as that associated with intestinal transit or tumor growth, can be interpreted by cells of preneoplastic colon tissue as a signal to initiate a -catenin dependent transcriptional program characteristic of cancer (10.2976/1.2955566)
    DOI : 10.2976/1.2955566
  • Ultrafast dynamics of ligands within heme proteins
    • Vos Marten H.
    Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics, Elsevier , 2008, 1777, pp.15-31 . Physiological bond formation and bond breaking events between proteins and ligands and their immediate consequences are difficult to synchronize and study in general. However, diatomic ligands can be photodissociated from heme, and thus in heme proteins ligand release and rebinding dynamics and trajectories have been studied on timescales of the internal vibrations of the protein that drive many biochemical reactions, and longer. The rapidly expanding number of characterized heme proteins involved in a large variety of functions allows comparative dynamics-structure-function studies. In this review, an overview is given of recent progress in this field, and in particular on initial sensing processes in signaling proteins, and on ligand and electron transfer dynamics in oxidases and cytochromes. (10.1016/j.bbabio.2007.10.004)
    DOI : 10.1016/j.bbabio.2007.10.004
  • Conformational changes in photoexcited (R)-(+)-1,1'-bi-2-naphthol studied by time-resolved circular dichroism
    • Niezborala Claire
    • Hache François
    Journal of the American Chemical Society, American Chemical Society , 2008, 130 (38), pp.12783-12786 . Conformational changes following photoexcitation of (R)-(+)-1,1′-bi-2-naphthol are studied with a time-resolved circular dichroism (CD) experiment. Two wavelengths are investigated. For λ = 237 nm, we observe a bleaching of the ground-state absorption and a transient CD structure. Thanks to a coupled-oscillator calculation, we can attribute this effect to a decrease of the dihedral angle. For λ = 245 nm, excited-state absorption and CD are observed. All these effects are solvent-dependent. In particular, it is shown that dynamics is slower in a protic solvent, which is attributed to hydrogen-bonding of the hydroxy groups with the solvent. (10.1021/ja8039844)
    DOI : 10.1021/ja8039844
  • Nanofluidics in the Debye Layer at Hydrophilic and Hydrophobic Surfaces
    • Bouzigues Cédric
    • Tabeling Paul
    • Bocquet L.
    Physical Review Letters, American Physical Society , 2008, 101, pp.114503 . By using evanescent waves, we study equilibrium and dynamical properties of liquid-solid interfaces in the Debye layer for hydrophilic and hydrophobic surfaces. We measure velocity profiles and nanotracer concentration and diffusion profiles between 20 and 300 nm from the walls in pressure-driven and electro-osmotic flows. We extract electrostatic and zeta potentials and determine hydrodynamic slip lengths with 10 nm accuracy. The spectacular amplification of the zeta potential resulting from hydrodynamic slippage allows us to clarify for the first time the dynamic origin of the zeta potential. (10.1103/PhysRevLett.101.114503)
    DOI : 10.1103/PhysRevLett.101.114503
  • Tissue deformation modulates Twist expression to determine anterior midgut differentiation in Drosophila embryos
    • Desprat Nicolas
    • Supatto Willy
    • Pouille Philippe-Alexandre
    • Beaurepaire Emmanuel
    • Farge Emmanuel
    Developmental Cell, Elsevier , 2008, 15 (3), pp.470-477 . Mechanical deformations associated with embryonic morphogenetic movements have been suggested to actively participate in the signaling cascades regulating developmental gene expression. Here we develop an appropriate experimental approach to ascertain the existence and the physiological relevance of this phenomenon. By combining the use of magnetic tweezers with in vivo laser ablation, we locally control physiologically relevant deformations in wild-type Drosophila embryonic tissues. We demonstrate that the deformations caused by germ band extension upregulate Twist expression in the stomodeal primordium. We find that stomodeal compression triggers Src42A-dependent nuclear translocation of Armadillo/β-catenin, which is required for Twist mechanical induction in the stomodeum. Finally, stomodeal-specific RNAi-mediated silencing of Twist during compression impairs the differentiation of midgut cells, resulting in larval lethality. These experiments show that mechanically induced Twist upregulation in stomodeal cells is necessary for subsequent midgut differentiation. DEVBIO (10.1016/j.devcel.2008.07.009)
    DOI : 10.1016/j.devcel.2008.07.009
  • Second Harmonic Microscopy to Quantify Renal Interstitial Fibrosis and Arterial Remodeling
    • Strupler Mathias
    • Hernest Monica
    • Fligny Cécile
    • Martin Jean-Louis
    • Tharaux Pierre-Louis
    • Schanne-Klein Marie-Claire
    Journal of Biomedical Optics, Society of Photo-optical Instrumentation Engineers , 2008, 13 (5), pp.054041 . Interstitial fibrosis is a powerful pejorative predictor of progression of nephropathies in a variety of chronic renal diseases. It is characterized by the depletion of kidney cells and their replacement by extracellular matrix, in particular, type-I fibrillar collagen, a protein scarce in normal interstitium. However, assessment of fibrosis remains a challenge in research and clinical pathology. We develop a novel methodology based on second harmonic generation SHG microscopy, and we image collagen fibers in human and mouse unstained kidneys. We take into account the variability in renal shape, and we develop automated image processing for quantitative scoring of thick murine tissues. This approach allows quantitative 3-D imaging of interstitial fibrosis and arterial remodeling with high accuracy. Moreover, SHG microscopy helps to raise pathophysiological questions. First, imaging of a large volume within a mouse kidney shows that progression of fibrosis is a heterogeneous process throughout the different renal compartments. Second, SHG from fibrillar collagens does not overlap with the glomerular tuft, despite patent clinical and experimental glomerulosclerosis. Since glomerulosclerosis involves SHG-silent nonfibrillar collagens, our work supports pathophysiological differences between interstitial fibrosis and glomerulosclerosis, a clearly nonfibrotic process. © 2008 Society of Photo-Optical Instrumentation Engineers (10.1117/1.2981830)
    DOI : 10.1117/1.2981830
  • Collective behavior during the exit of a wetting liquid through a network of channels
    • Baroud Charles N.
    • Wang Xin C.
    • Masson Jean-Baptiste
    Journal of Colloid and Interface Science, Elsevier , 2008, 326 (2), pp.445 . The exit of a wetting fluid from a thin microchannel into a sudden expansion is studied experimentally. In the case of the exit from a single channel, the advancing interface converges to a parabolic shape after an initial transient, in accordance with the lubrication limit analysis of a spreading drop. The experiments are then repeated for the exit from two parallel channels. At early times, the two exiting drops behave independently and display the same evolution as a single exiting droplet, while at late times we recover a single parabolic profile. The transition between the early and late states is due to the merging of the two drops, which is associated with a sudden increase in the flow rate. This is the signature of a collective effect which acts to redistribute the fluid spatially. Finally, the experiment is generalized to the case of seven parallel channels where a cascade of two-by-two mergings is observed, indicating that local interactions dominate the dynamics which lead to the global state of the system. Cop. 2008 Elsevier Inc. All rights reserved. (10.1016/j.jcis.2008.06.040)
    DOI : 10.1016/j.jcis.2008.06.040
  • Flavin-dependent thymidylate synthase X limits chromosomal DNA replication
    • Escartin Frédéric
    • Skouloubris Stéphane
    • Liebl Ursula
    • Myllykallio Hannu
    Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2008, 105 (29), pp.9948-9952 .
  • Electron hopping through the 15 Å triple tryptophan molecular wire in DNA photolyase occurs within 30 ps
    • Lukacs Andras
    • Eker André P.M.
    • Byrdin Martin
    • Brettel Klaus
    • Vos Marten H.
    Journal of the American Chemical Society, American Chemical Society , 2008, 130 (44), pp.14394 . DNA photolyase is a photoactive flavoprotein that contains three tryptophan residues between the FAD cofactor and the protein surface, the solvent-exposed Trp being located 14.8 Å from the flavin. Photoreduction of the neutral radical FADH* form to the catalytically active FADH- form occurs via electron transfer through this chain. The first step in this chain takes 30 ps, the second less than 4 ps. Using a combination of site-directed mutagenesis and femtosecond polarization spectroscopy to discriminate the spectroscopically indistinguishable Trp residues, we show that the third step occurs in less than 30 ps. This implies that the first photoreduction step is rate limiting and that the Trp chain effectively acts as molecular "wire" ensuring rapid and directed long-range charge translocation across the protein. This finding is important for the functioning of the large class of cryptochrome blue-light receptors, where the Trp chain is conserved. In DNA photolyase we make use of the natural photoactivation of the process, but more generally chains of aromatic amino acids may allow very fast long-range electron transfer also in nonphotoactive proteins. Copyright Cop. 2008 American Chemical Society. (10.1021/ja805261m)
    DOI : 10.1021/ja805261m
  • Third-harmonic generation microscopy with focus-engineered beams: a numerical study
    • Olivier Nicolas
    • Beaurepaire Emmanuel
    Optics Express, Optical Society of America - OSA Publishing , 2008, 16 (19), pp.14703-14715 . We use a vector field model to analyze third-harmonic generation (THG) from model geometries (interfaces, slabs, periodic structures) illuminated by Hermite-Gaussian (HG) and Laguerre-Gaussian (LG) beams focused by a high NA lens. Calculations show that phase matching conditions are significantly affected by the tailoring of the field distribution near focus. In the case of an interface parallel to the optical axis illuminated by an odd HG mode, the emission patterns and signal level reflect the relative orientation of the interface and the focal field structure. In the case of slabs and periodic structures, the emission patterns reflect the interplay between focal field distribution (amplitude and phase) and sample structure. Forward-to-backward emission ratios using different beam shapes provide sub-wavelength information about sample spatial frequencies. © 2008 Optical Society of America (10.1364/OE.16.014703)
    DOI : 10.1364/OE.16.014703
  • Organic functionalization of luminescent oxide nanoparticles towards their application as biological probes
    • Giaume Domitille
    • Poggi Mélanie
    • Casanova Didier
    • Mialon Geneviève
    • Lahlil Khalid
    • Alexandrou Antigoni
    • Gacoin Thierry
    • Boilot Jean-Pierre
    Langmuir, American Chemical Society , 2008, 24 (19), pp.11018 . Luminescent inorganic nanoparticles are now widely studied for their applications as biological probes for in vitro or in vivo experiments. The functionalization of the particles is a key step toward these applications, since it determines the control of the coupling between the particles and the biological species of interest. This paper is devoted to the case of rare earth doped oxide nanoparticles and their functionalization through their surface encapsulation with a functional polysiloxane shell. The first step of the process is the adsorption of silicate ions that will act as a primary layer for the further surface polymerization of the silane, either aminopropyltriethoxysilane (APTES) or glycidoxypropyltrimethoxysilane (GPTMS). The amino- or epoxy- functions born by the silane allow the versatile coupling of the particles with bio-organic species following the chemistry that is commonly used in biochips. Special attention is paid to the careful characterization of each step of the functionalization process, especially concerning the average number of organic functions that are available for the final coupling of the particles with proteins. The surface density of amino or epoxy functions was found to be 0.4 and 1.9 functions per square nanometer for GPTMS and APTES silanized particles, respectively. An example of application of the amino-functionalized particles is given for the coupling with α-bungarotoxins. The average number (up to 8) and the distribution of the number of proteins per particle are given, showing the potentialities of the functionalization process for the labeling of biological species. (10.1021/la8015468)
    DOI : 10.1021/la8015468
  • Low-loss polymers for terahertz applications
    • Podzorov Alexander
    • Gallot Guilhem
    Applied optics, Optical Society of America , 2008, 47, pp.3254 .
  • Anomalies in the disappearance of the extraordinary electromagnetic transmission in subwavelength hole arrays
    • Masson Jean-Baptiste
    • Podzorov Alexander
    • Gallot Guilhem
    Optics Express, Optical Society of America - OSA Publishing , 2008, 16, pp.4719 .
  • Flavin-dependent thymidylate synthase ThyX activity: implications for the folate cycle in bacteria.
    • Leduc Damien
    • Escartin Frédéric
    • Nijhout H Frederik
    • Reed Michael C
    • Liebl Ursula
    • Skouloubris Stéphane
    • Myllykallio Hannu
    Journal of Bacteriology, American Society for Microbiology , 2007, 189 (23), pp.8537-45 . Although flavin-dependent ThyX proteins show thymidylate synthase activity in vitro and functionally complement thyA defects in heterologous systems, direct proof of their cellular functions is missing. Using insertional mutagenesis of Rhodobacter capsulatus thyX, we constructed the first defined thyX inactivation mutant. Phenotypic analyses of the obtained mutant strain confirmed that R. capsulatus ThyX is required for de novo thymidylate synthesis. Full complementation of the R. capsulatus thyX::spec strain to thymidine prototrophy required not only the canonical thymidylate synthase ThyA but also the dihydrofolate reductase FolA. Strikingly, we also found that addition of exogenous methylenetetrahydrofolate transiently inhibited the growth of the different Rhodobacter strains used in this work. To rationalize these experimental results, we used a mathematical model of bacterial folate metabolism. This model suggests that a very low dihydrofolate reductase activity is enough to rescue significant thymidylate synthesis in the presence of ThyX proteins and is in agreement with the notion that intracellular accumulation of folates results in growth inhibition. In addition, our observations suggest that the presence of flavin-dependent thymidylate synthase X provides growth benefits under conditions in which the level of reduced folate derivatives is compromised. (10.1128/JB.01380-07)
    DOI : 10.1128/JB.01380-07
  • Signal epidetection in third-harmonic generation microscopy of turbid media
    • Débarre Delphine
    • Olivier Nicolas
    • Beaurepaire Emmanuel
    Optics Express, Optical Society of America - OSA Publishing , 2007, 15, pp.8913-8924 . Third-harmonic generation (THG) imaging of thick samples or large organisms requires TH light to be epicollected through the focusing objective. In this study we first estimate the amount of backward-to-forward TH radiation created by an isolated object as a function of size and spatial frequencies in the object. Theory and model experiments indicate that no significant signal can be epidetected from a (biological) dielectric structure embedded in a transparent medium. In contrast, backward emission is observed from metal nanoparticles where THG is partly a surface effect. We then address the case of an object embedded in a turbid medium. Experiments and Monte Carlo simulations show that epidetection is possible when the absorption mean free path of harmonic light in the medium exceeds its reduced scattering length, and that epicollection efficiency critically depends on the microscope field-of-view even at shallow depths, because backscattered light is essentially diffusive. These observations provide guidelines for optimizing epidetection in third-harmonic, second-harmonic, or CARS imaging of thick tissues. (10.1364/OE.15.008913)
    DOI : 10.1364/OE.15.008913
  • A NADPH substitute for selective photo-initiation of reductive bioprocesses via two-photon induced electron transfer.
    • Robin Anne-Claire
    • Gmouh Saïd
    • Mongin Olivier
    • Jouikov Viatcheslav
    • Werts Martinus
    • Gautier Clément
    • Slama-Schwok Anny
    • Blanchard Desce Mireille
    Chemical Communications, Royal Society of Chemistry , 2007 (13), pp.1334-6 . A NADPH substitute where the nicotinamide moiety is replaced by a chromophoric unit having much larger two-photon absorption cross-section and able to transfer electrons to flavins only upon excitation is described as an effective two-photon nanotrigger for selective photo-activation of electron transfer in bioreductive processes. (10.1039/b615628b)
    DOI : 10.1039/b615628b
  • Second harmonic imaging and scoring of collagen in fibrotic tissues.
    • Strupler Mathias
    • Pena Ana-Maria
    • Hernest Monica
    • Tharaux Pierre-Louis
    • Martin Jean-Louis
    • Beaurepaire Emmanuel
    • Schanne-Klein Marie-Claire
    Optics Express, Optical Society of America - OSA Publishing , 2007, 15 (7), pp.4054-4065 . We compare second harmonic generation (SHG) to histological and immunohistochemical techniques for the visualization and scoring of collagen in biological tissues. We show that SHG microscopy is highly specific for fibrillar collagens and that combined SHG and two-photon excited fluorescence (2PEF) imaging can provide simultaneous three-dimensional visualization of collagen synthesis and assembly sites in transgenic animal models expressing GFP constructs. Finally, we propose several scores for characterizing collagen accumulation based on SHG images and appropriate for different types of collagen distributions. We illustrate the sensitivity of these scores in a murine model of renal fibrosis using a morphological segmentation of the tissue based on endogenous 2PEF signals. © 2007 Optical Society of America (10.1364/OE.15.004054)
    DOI : 10.1364/OE.15.004054
  • Synchronous photoinitiation of endothelial NO synthase activity by a nanotrigger targeted as its NADPH site
    • Beaumont Edward
    • Lambry Jean-Christophe
    • Gautier Clément
    • Robin Anne-Claire
    • Gmouh Saïd
    • Berka Vladimir
    • Tsai Ah-Lim
    • Blanchard Desce Mireille
    • Slama-Schwok Anny
    Journal of the American Chemical Society, American Chemical Society , 2007, 129 (7), pp.2178-2186 . We designed a new nanotrigger to synchronize and monitor an enzymatic activity interacting specifically with the conserved NADPH binding site. The nanotrigger (NT) combines a docking moiety targeting the NADPH site and a chromophore moiety responsive to light excitation for efficient electron transfer to the protein. Specific binding of the nanotrigger to the reductase domain of the endothelial nitric oxide synthase (eNOSred) was demonstrated by competition between NADPH and the nanotrigger on the reduction of eNOSred flavin. A micromolar Ki was estimated. We had monitored initiation of eNOSred activity by ultrafast transient spectroscopy. The transient absorption spectrum recorded at 250 ps fits the expected sum of the reduced and oxidized species, independently obtained by other chemical methods, in agreement with a photoinduced electron transfer from the excited nanotrigger to the flavin moiety of eNOSred. The rate of electron transfer from the excited state of the nanotrigger (NT*) to the protein is estimated to be kET = (7 ± 2) × 109 s-1 using the decay of oxidized eNOSred-bound nanotrigger compared against prereduced eNOSred or glucose 6-P dehydrogenase as controls. This fast electron transfer bypasses the slow hydride transfer to initiate NOS catalysis as shown by ultrafast kinetics using the eNOSred mutated in the regulatory F1160 residue. The selective targeting of the nanotrigger to NADPH sites should allow controlled initiation of the enzymatic activity of numerous proteins containing an NADPH site. (10.1021/ja067543e)
    DOI : 10.1021/ja067543e
  • Quantitative characterization of biological liquids for third-harmonic generation microscopy.
    • Débarre Delphine
    • Beaurepaire Emmanuel
    Biophysical Journal, Biophysical Society , 2007, 92 (2), pp.603-12 . Third-harmonic generation (THG) microscopy provides images of unstained biological samples based on spatial variations in third-order nonlinear susceptibility, refractive index, and dispersion. In this study, we establish quantitative values for the third-order nonlinear susceptibilities of several solvents (water, ethanol, glycerol), physiological aqueous (ions, amino acids, polypeptides, bovine serum albumin, glucose) and lipid (triglycerides, cholesterol) solutions as a function of solute concentration in the 1.05-1.25 microm excitation range. We use these data in conjunction with imaging experiments to show that THG imaging with approximately 1.2 microm excitation lacks specificity and sensitivity to detect physiological ion concentration changes, and that nonaqueous structures such as lipid bodies provide a more robust source of signal. Finally, we illustrate the impact of index-matching liquids in THG images. These data provide a basis for interpreting biological THG images and for developing additional applications. (10.1529/biophysj.106.094946)
    DOI : 10.1529/biophysj.106.094946
  • Ligand dynamics in an electron-transfer protein: picosecond geminate recombination of carbon monoxide to heme in mutant forms of cytochrome c
    • Silkstone Gary
    • Jasaitis Audrius
    • Wilson M. T.
    • Vos Marten H.
    Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2007, 282, pp.1638-1649 . Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to ∼70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO. (10.1074/jbc.M605760200)
    DOI : 10.1074/jbc.M605760200
  • Distal Val346Ile mutation in inducible NO synthase promotes substrate-dependent NO confinement
    • Beaumont Edward
    • Lambry Jean-Christophe
    • Wang Z.-Q.
    • Stuehr D.J.
    • Martin Jean-Louis
    • Slama-Schwok Anny
    Biochemistry, American Chemical Society , 2007, 46 (47), pp.13533 . The function of inducible NO synthase (WT iNOS) depends on the release of NO from the ferric heme before the enzyme is reduced. Key parameters controlling ligand dynamics include the distal and proximal heme pocket amino acids, as well as the inner solvent molecules. In this work, we tested how a point mutation in the distal heme side of WT iNOS affected the geminate rebinding of NO by ultrafast kinetics and molecular dynamics simulations. The mutation sequestered much of the photodissociated NO close to the heme compared to WT iNOS, with a main picosecond phase accounting for 78% of the rebinding to the arginine-bound Val346Ile protein. Consequently, the probability of NO release from Val346Ile decreased as compared to that from WT iNOS, provided the substrate binding site is filled. These data are rationalized by a steric effect of the Ile methyl group inducing events mediated by the substrate, transmitted via the propionates to the NO and the protein. This model is consistent with the role of the H-bonding network involving the heme, the substrate, and the BH4 cofactor in controlling NO release, with a key role of the heme propionates [Gautier et al. (2006) Nitric Oxide 15, 312]. These data support the effect of Val346Ile mutation in decreasing NO release and slowing down NO synthesis compared to WT iNOS determined by single turnover catalysis [Wang et al. (2004) J. Biol. Chem. 279, 19018]. Cop. 2007 American Chemical Society. (10.1021/bi701567h)
    DOI : 10.1021/bi701567h
  • Effects of lindane (gamma-hexachlorocyclohexane) on rat heart muscle contraction.
    • Sauviat Martin-Pierre
    • Pagès Nicole
    General Physiology and Biophysics, Rand d Print , 2007, 26 (4), pp.275 . The effects of micromolar concentrations of lindane on the mechanical activity of cardiac left ventricular papillary muscles were studied in adult female rats. Lindane decreased the amplitude and duration of the contraction, and slowed down the time course of its ascending phase (i.e. decreased the maximum rate of rise of the initial phase (dC/ dtmax)). Both amplitude and duration of the contraction, but not dC/dtmax, were restored by subsequent application of the rapid delayed outward K+ current (IKr) blocker E-4031 (10 nmol/l). Increasing the stimulation frequency from 1 to 3.3 Hz in the control solution produced a decrease in the amplitude of the first beat peak contraction while a slow recovery phase (srp) developed, as the result of the Na+-Ca2+ exchanger activity. When the frequency was restored to 1 Hz, a post rest potentiation (prp) with a negative staircase (ns) developed due to the sarcoplasmic reticulum (SR) Ca2+ refilling. Lindane increased the amplitude of both srp and prp, but did not affect ns, which indicates that SR Ca2+ refilling was not altered by the pesticide. In conclusion, the results strongly suggest that some of the lindane-induced negative inotropic and chronotropic-like effects on the contraction are due to an increased IKr while the decrease in dC/dtmax (i.e. the rate of cross-bridge formation) results from lindane oxidative properties.