Articles

Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates
- Botcherby Edward J.
- Smith Christopher W.
- Kohl Michael
- Débarre Delphine
- Booth Martin J.
- Juskaitis Rimas
- Paulsen Ole
- Wilson Tony
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2011, 109 (8), pp.2919-2924 . Multiphoton microscopy is a powerful tool in neuroscience, promising to deliver important data on the spatiotemporal activity within individual neurons as well as in networks of neurons. A major limitation of current technologies is the relatively slow scan rates along the z direction compared to the kHz rates obtainable in the x and y directions. Here,we describe a custom-built microscope system based on an architecture that allows kHz scan rates over hundreds of microns in all three dimensions without introducing aberration. We further demonstrate how this high-speed 3D multiphoton imaging system can be used to study neuronal activity at millisecond resolution at the subcellular as well as the population level. (10.1073/pnas.1111662109)
DOI : 10.1073/pnas.1111662109
Structure and function of a novel endonuclease acting on branched DNA substrates
- Creze C.
- Lestini Roxane
- Kühn Joëlle
- Ligabue Alessio
- Becker Hubert F.
- Czjzek M.
- Flament D.
- Myllykallio Hannu
Biochemical Society Transactions, Portland Press , 2011, 39 (1), pp.145-149 . Branched DNA structures that occur during DNA repair and recombination must be efficiently processed by structure-specific endonucleases in order to avoid cell death. In the present paper, we summarize our screen for new interaction partners for the archaeal replication clamp that led to the functional characterization of a novel endonuclease family, dubbed NucS. Structural analyses of Pyrococcus abyssi NucS revealed an unexpected binding site for ssDNA (single-stranded DNA) that directs, together with the replication clamp, the nuclease activity of this protein towards ssDNA-dsDNA (double-stranded DNA) junctions. Our studies suggest that understanding the detailed architecture and dynamic behaviour of the NucS (nuclease specific for ssDNA)-PCNA (proliferating-cell nuclear antigen) complex with DNA will be crucial for identification of its physiologically relevant activities. ©The Authors Journal compilation ©2011 Biochemical Society. (10.1042/BST0390145)
DOI : 10.1042/BST0390145
Few femtosecond, few kiloampere electron bunch produced by a laser-plasma accelerator
- Lundh Olle
- Lim J.
- Rechatin Clément
- Ammoura L.
- Ben-Ismaïl Ahmed
- Davoine X.
- Gallot Guilhem
- Goddet Jean-Philippe
- Lefebvre E.
- Malka Victor
- Faure Jérôme
Nature Physics, Nature Publishing Group [2005-....] , 2011, 7 (3), pp.219 . Particle accelerators driven by the interaction of ultraintense and ultrashort laser pulses with a plasma(1) can generate accelerating electric fields of several hundred gigavolts per metre and deliver high-quality electron beams with low energy spread(2-5), low emittance(6) and up to 1 GeV peak energy(7,8). Moreover, it is expected they may soon be able to produce bursts of electrons shorter than those produced by conventional particle accelerators, down to femtosecond durations and less. Here we present wide-band spectral measurements of coherent transition radiation which we use for temporal characterization. Our analysis shows that the electron beam, produced using controlled optical injection(9), contains a temporal feature that can be identified as a 15 pC, 1.4-1.8 fs electron bunch (root mean square) leading to a peak current of 3-4 kA depending on the bunch shape. We anticipate that these results will have a strong impact on emerging applications such as short-pulse and short-wavelength radiation sources(10,11), and will benefit the realization of laboratory-scale free-electron lasers(12-14). (10.1038/NPHYS1872)
DOI : 10.1038/NPHYS1872
Sub-picosecond Raman spectrometer for time-resolved studies of structural dynamics in heme proteins
- Kruglik Sergei G.
- Lambry Jean-Christophe
- Martin Jean-Louis
- Vos Marten H.
- Négrerie Michel
Journal of Raman Spectroscopy, Wiley , 2011, 42 (3), pp.265 . We describe a pump-probe Raman spectrometer based on a femtosecond Ti:sapphire laser, an optical parametric generator and two optical parametric amplifiers for time-resolved studies, with emphasis on the structural dynamics in heme proteins. The system provides a 100-fs pump pulse tunable in the range 500-600 nm and a transform-limited sub-picosecond probe pulse tunable in the range 390-450 nm. The spectrometer has spectral (25 cm(-1)) and temporal (similar to 0.7 ps) resolutions which constitute an effective compromise for identifying transient heme protein species and for following their structural evolution by spontaneous Raman scattering in the time range 0.5 ps to 2 ns. This apparatus was applied to time-resolved studies of a broad range of heme proteins, monitoring the primary dynamics of photoinduced heme coordination state and structural changes, its interaction with protein side-chains and diatomic gaseous ligands, as well as heme vibrational cooling. The treatment of transient Raman spectra is described in detail, and the advantages and shortcomings of spontaneous resonance Raman spectroscopy for ultrafast heme proteins studies are discussed. We demonstrate the efficiency of the constructed spectrometer by measuring Raman spectra in the sub-picosecond and picosecond time ranges for the oxygen-storage heme protein myoglobin and for the oxygen-sensor heme protein FixLH in interaction with the diatomic gaseous ligands CO, NO, and O-2. Copyright (C) 2010 John Wiley and Sons, Ltd. (10.1002/jrs.2685)
DOI : 10.1002/jrs.2685
Deep and fast live imaging with two-photon scanned light-sheet microscopy
- Truong T.V.
- Supatto Willy
- Koos D.S.
- Choi J.M.
- Fraser S.E.
Nature Methods, Nature Publishing Group , 2011, 8 (9), pp.757 . We implemented two-photon scanned light-sheet microscopy, combining nonlinear excitation with orthogonal illumination of light-sheet microscopy, and showed its excellent performance for in vivo, cellular-resolution, three-dimensional imaging of large biological samples. Live imaging of fruit fly and zebrafish embryos confirmed that the technique can be used to image up to twice deeper than with one-photon light-sheet microscopy and more than ten times faster than with point-scanning two-photon microscopy without compromising normal biology. Cop. 2011 Nature America, Inc. All rights reserved. (10.1038/nmeth.1652)
DOI : 10.1038/nmeth.1652
Filming atomic motions in liquids
- Bratos S.
- Gallot Guilhem
- Leicknam J.-C.
- Pommeret Stanislas
- Vuilleumier Rodolphe
- Wulff Michael
Acta Chimica Slovenica, Slovenian Chemical Society , 2011, 58 (3), pp.434 . Basic techniques in ultrafast time-resolved optical spectroscopy and x-ray diffraction are described for a broad scientific community. Basic experimental setups are presented, and theories for the interpretation of experimental data are briefly described. The power of these ultrafast techniques is shown with a few selected examples. It is shown in particular how they permit to film atomic motions during a chemical reaction. The strong and weak points of the two complementary techniques are discussed in some detail. A number of basic references are included to help interested readers. Future developments of ultrafast techniques are conjectured at the end of the paper.
Brewster's angle silicon wafer terahertz linear polarizer
- Wojdyla Antoine
- Gallot Guilhem
Optics Express, Optical Society of America - OSA Publishing , 2011, 19 (15), pp.14099 . We present a new cost-effective terahertz linear polarizer made from a stack of silicon wafers at Brewster's angle, andevaluate its performances. We show that this polarizer is wide-band, has a high extinction ratio (> 6 × 103) and very small insertion losses (inf 1%). We provide measurements of the temporal waveforms after linearly polarizing the THz beam and show that there is no distortion of the pulse. We compare its performances with a commercial wire-grid polarizer, and show that the Brewster's angle polarizer can conveniently be used to control the power of a terahertz beam. Cop. 2011 Optical Society of America. (10.1364/OE.19.014099)
DOI : 10.1364/OE.19.014099
Rails and anchors: Guiding and trapping droplet microreactors in two dimensions
- Abbyad Paul
- Dangla Rémi
- Alexandrou Antigoni
- Baroud Charles N.
Lab on a Chip, Royal Society of Chemistry , 2011, 11 (5), pp.813-821 . This paper presents a method to control the motion of nanolitre drops in a wide and thin microchannel, by etching fine patterns into the channel's top surface. Such control is possible for drops that are squeezed by the channel roof, by allowing them to reduce their surface energy as they enter into a local depression. The resulting gain in surface energy pulls a drop into the groove such that localized holes can be used as anchors for holding drops, while linear patterns can be used as rails to guide them along complex trajectories. An anchored drop can remain stationary indefinitely, as long as the driving flow rate is below a critical value which depends on the hole and drop sizes. By micro-fabricating holes into a grid pattern, drops can be arrayed and held in the observation field of a microscope against the mean carrier flow. Their contents can then be modulated by gas exchange with the flowing carrier oil. We demonstrate in particular how the pH or the oxygen levels within the drops can be controlled spatially and temporally, either by exposing rows of drops to two streams of oil at different gas concentrations or by periodically switching oil inputs to vary the gas concentration of drops as a function of time. Oxygen control is used to selectively deoxygenate droplets that encapsulate red blood cells from patients suffering from sickle cell disease, in order to study the polymerization of intracellular hemoglobin. Cycles of oxygenation and deoxygenation of anchored droplets induce depolymerization and polymerization of the hemoglobin, thus providing a method to simulate the cycling that takes place in physiological flows. Cop. 2011 The Royal Society of Chemistry. (10.1039/c0lc00104j)
DOI : 10.1039/c0lc00104j
Dynamics of NO interacting with soluble guanylate cyclase from 1 ps to 0.1 s and induced structural transitions
- Yoo Byung-Kuk
- Lamarre Isabelle
- Martin Jean-Louis
- Rappaport Fabrice
- Negrerie Michel
BMC Pharmacology, BioMed Central , 2011, 11 (Suppl 1), pp.P77 .
Combining rails and anchors with laser forcing for selective manipulation within 2D droplet arrays.
- Fradet Etienne
- Mcdougall Craig
- Abbyad Paul
- Dangla Rémi
- Mcgloin David
- Baroud Charles N.
Lab on a Chip, Royal Society of Chemistry , 2011, 11 (24), pp.4228-4234 . We demonstrate the combination of a rails and anchors microfluidic system with laser forcing to enable the creation of highly controllable 2D droplet arrays. Water droplets residing in an oil phase can be pinned to anchor holes made in the base of a microfluidic channel, enabling the creation of arrays by the appropriate patterning of such holes. The introduction of laser forcing, via laser induced thermocapillary forces to anchored droplets, enables the selective extraction of particular droplets from an array. We also demonstrate that such anchor arrays can be filled with multiple, in our case two, droplets each and that if such droplets have different chemical contents, the application of a laser at their interface triggers their merging and a chemical reaction to take place. Finally by adding guiding rails within the microfluidic structure we can selectively fill large scale arrays with monodisperse droplets with significant control over their contents. In this way we make a droplet array filled with 96 droplets containing different concentrations of fluorescent microparticles. (10.1039/C1LC20541B)
DOI : 10.1039/C1LC20541B
Vibrational Motions Associated with Primary Processes in Bacteriorhodopsin Studied by Coherent Infrared Emission Spectroscopy
- Groma Geza I.
- Colonna Anne
- Martin Jean-Louis
- Vos Marten H.
Biophysical Journal, Biophysical Society , 2011, 100 (6), pp.1578 . The primary energetic processes driving the functional proton pump of bacteriorhodopsin take place in the form of complex molecular dynamic events after excitation of the retinal chromophore into the Franck-Condon state. These early events include a strong electronic polarization, skeletal stretching, and all-trans-to-13-cis isomerization upon formation of the J intermediate. The effectiveness of the photoreaction is ensured by a conical intersection between the electronic excited and ground states, providing highly nonadiabatic coupling to nuclear motions. Here, we study real-time vibrational coherences associated with these motions by analyzing light-induced infrared emission from oriented purple membranes in the 750-1400 cm(-1) region. The experimental technique applied is based on second-order femtosecond difference frequency generation on macroscopically ordered samples that also yield information on phase and direction of the underlying motions. Concerted use of several analysis methods resulted in the isolation and characterization of seven different vibrational modes assigned as C-C stretches, out-of-plane methyl rocks, and hydrogen out-of-plane wags, whereas no in-plane H rock was found. Based on their lifetimes and several other criteria, we deduce that the majority of the observed modes take place on the potential energy surface of the excited electronic state. In particular, the direction sensitivity provides experimental evidence for large intermediate distortions of the retinal plane during the excited-state isomerization process. (10.1016/j.bpj.2011.02.011)
DOI : 10.1016/j.bpj.2011.02.011
Strong Ligand-Protein Interactions Revealed by Ultrafast Infrared Spectroscopy of CO in the Heme Pocket of the Oxygen Sensor FixL
- Nuernberger Patrick
- Lee Kevin F.
- Bonvalet Adeline
- Bouzhir-Sima Latifa
- Lambry Jean-Christophe
- Liebl Ursula
- Joffre Manuel
- Vos Marten H.
Journal of the American Chemical Society, American Chemical Society , 2011, 133 (43), pp.17110 . In heme-based sensor proteins, ligand binding to heme in a sensor domain induces conformational changes that eventually lead to changes in enzymatic activity of an associated catalytic domain. The bacterial oxygen sensor FixL is the best-studied example of these proteins and displays marked differences in dynamic behavior with respect to model globin proteins. We report a mid-IR study of the configuration and ultrafast dynamics of CO in the distal heme pocket site of the sensor PAS domain FixLH, employing a recently developed method that provides a unique combination of high spectral resolution and range and high sensitivity. Anisotropy measurements indicate that CO rotates toward the heme plane upon dissociation, as is the case in globins. Remarkably, CO bound to the heme iron is tilted by similar to 30 degrees with respect to the heme normal, which contrasts to the situation in myoglobin and in present FixLH-CO X-ray crystal structure models. This implies protein-environment-induced strain on the ligand, which is possibly at the origin of a very rapid docking-site population in a single conformation. Our observations likely explain the unusually low affinity of FixL for CO that is at the origin of the weak ligand discrimination between CO and O(2). Moreover, we observe orders of magnitude faster vibrational relaxation of dissociated CO in FixL than in globins, implying strong interactions of the ligand with the distal heme pocket environment. Finally, in the R220H FixLH mutant protein, where CO is H-bonded to a distal histidine, we demonstrate that the H-bond is maintained during photolysis. Comparison with extensively studied globin proteins unveils a surprisingly rich variety in both structural and dynamic properties of the interaction of a diatomic ligand with the ubiquitous b-type heme-proximal histidine system in different distal pockets. (10.1021/ja204549n)
DOI : 10.1021/ja204549n
Measurement of circular dichroism dynamics in a nanosecond temperature-jump experiment
- Khuc Mai-Thu
- Mendonça Lucille
- Sharma S.
- Volk M.
- Solinas Xavier
- Hache François
Review of Scientific Instruments, American Institute of Physics , 2011, 82 (5) . The use of a fast temperature jump (T-jump) is a very powerful experiment aiming at studying protein denaturation dynamics. However, probing the secondary structure is a difficult challenge and rarely yields quantitative values. We present the technical implementation of far-UV circular dichroism in a nanosecond T-jump experiment and show that this experiment allows us to follow quantitatively the change in the helical fraction of a poly(glutamic acid) peptide during its thermal denaturation with 12 ns time resolution. Cop. 2011 American Institute of Physics. (10.1063/1.3592331)
DOI : 10.1063/1.3592331
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
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
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
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
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
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
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
A mechanism for the polarity formation of chemoreceptors at the growth cone membrane for gradient amplification during directional sensing
- Bouzigues Cédric
- Holcman D.
- Dahan Maxime
PLoS ONE, Public Library of Science , 2010, 5 (2) . Accurate response to external directional signals is essential for many physiological functions such as chemotaxis or axonal guidance. It relies on the detection and amplification of gradients of chemical cues, which, in eukaryotic cells, involves the asymmetric relocalization of signaling molecules. How molecular events coordinate to induce a polarity at the cell level remains however poorly understood, particularly for nerve chemotaxis. Here, we propose a model, inspired by single-molecule experiments, for the membrane dynamics of GABA chemoreceptors in nerve growth cones (GCs) during directional sensing. In our model, transient interactions between the receptors and the microtubules, coupled to GABA-induced signaling, provide a positive-feedback loop that leads to redistribution of the receptors towards the gradient source. Using numerical simulations with parameters derived from experiments, we find that the kinetics of polarization and the steady-state polarized distribution of GABA receptors are in remarkable agreement with experimental observations. Furthermore, we make predictions on the properties of the GC seen as a sensing, amplification and filtering module. In particular, the growth cone acts as a low-pass filter with a time constant -10 minutes determined by the Brownian diffusion of chemoreceptors in the membrane. This filtering makes the gradient amplification resistent to rapid fluctuations of the external signals, a beneficial feature to enhance the accuracy of neuronal wiring. Since the model is based on minimal assumptions on the receptor/cytoskeleton interactions, its validity extends to polarity formation beyond the case of GABA gradient sensing. Altogether, it constitutes an original positive-feedback mechanism by which cells can dynamically adapt their internal organization to external signals. Cop. 2010 Bouzigues et al. (10.1371/journal.pone.0009243)
DOI : 10.1371/journal.pone.0009243