Publications

2005

• Source à rayons X compacte
  
  • Bonvalet Adeline
  • Martin Jean-Louis
  • Audebert Patrick
  • Sintes Jean-Marc
  • Darmon Adeline
  , 2005.
• Analogues photoactivables du NADH, du NADPH, du NAD+ ou du NADP+
  
  • Slama-Schwok Anny
  • Blanchard Desce Mireille
  • Gmouh Saïd
  • Gautier Clément
  • Martin Jean-Louis
  , 2005. invention concerne des analogues photoactivables du NADH, du NADPH, du NAD+ ou du NADP+ permettant de déclencher de façon sélective une séquence enzymatique précise et de ce fait de suivre par spectroscopie optique rapide le fonctionnement de la machinerie enzymatique.
• Dynamique conformationnelle de la myoglobine suivie par dichroïsme circulaire résolu temporellement.
  
  • Dartigalongue Thibault
  , 2005. L'objet de ce travail de thèse est l'étude de la photodissociation de la carboxymyoglobine (MbCO) par dichroïsme circulaire résolu temporellement (TRCD). La myoglobine (Mb) est une protéine responsable du stockage de l'oxygène dans les muscles de l'organisme. Lorsque le complexe MbCO absorbe un photon visible, il y a photodissociation, le CO se détache et quitte la protéine. Il s'agit donc de mesurer le dichroïsme circulaire de la myoglobine juste après la photodissociation, afin de pouvoir remonter aux changements de structure ultra rapides de la protéine. D'un point de vue expérimental, la difficulté de ce travail résidait dans la maîtrise des nombreux artefacts d'une telle expérience. D'un point de vue théorique, un calcul basé sur la théorie de la polarisabilité a permis de montre! r que le CD était sensible essentiellement aux mouvements de l'histidine proximale, résidu clef dans la compréhension de l'effet allostérique. Le résultat marquant de cette étude a donc été la mise en évidence d'une dynamique temporelle de 100 ps, attribuée à un mouvement de l'histidine proximale. Une nouvelle source laser a également été développée dans le but de faire des expériences de TRCD dans l'ultraviolet. L'objectif est de pouvoir suivre à terme la dynamique de repliement des protéines. Des résultats préliminaires ont été obtenus.
• Polarisation ultrarapide et mouvements vibrationnels dans la bactériorhodopsine étudiés par spectroscopie cohérente d'émission infrarouge.
  
  • Colonna Anne
  , 2005. Ce travail, qui se place dans le cadre général de l'étude de la dynamique primaire des protéines, concerne les protéines à rétinal, protéines impliquées en particulier dans la vision. La protéine bactérienne modèle utilisée est la bactériorhodopsine, dont le rôle est la conversion de l'énergie lumineuse en un gradient de protons. L'interaction photon-rétinal induit initialement deux phénomènes, dont le rôle et la chronologie sont encore incertains: isomérisation du rétinal en quelques centaines de femtosecondes et établissement d'une polarisation ultrarapide au niveau du système rétinal/protéine. La méthode spectroscopique femtoseconde utilisée permet de détecter et quantifier des déplacements de charges avec une résolution temporelle de 13 fs, et ainsi de séparer temporellement ces deux phénomènes. Elle est basée sur un phénomène non linéaire du deuxième ordre, le redressement optique: l'excitation avec un champ électrique oscillant d'un matériau non centrosymétrique va créer une polarisation pointant toujours dans le même sens. Ce matériau consiste de multicouches anhydres orientées de membranes pourpres contenant la bactériohodopsine. Le champ infrarouge émis suite à l'établissement de cette polarisation est détecté en le faisant interférer avec un signal infrarouge de référence, généré par redressement optique dans un cristal non linéaire (GaAs ou AgGaS2). Le champ infrarouge émis par la bactériorhodopsine reflète directement la différence entre les moments dipolaires de l'état fondamental et de l'état excité. En plus de la réponse électronique instantanée de l'échantillon, une émission infrarouge est détectée qui dure sur plusieurs picosecondes, caractéristique des mouvements de charges associés aux mouvements vibrationnels du système rétinal/protéine. La séparation des deux types de réponse et la description d'etaillée, en fréquence et en phase, de l'ensemble du signal complexe nécessitent l'utilisation concertée de plusieurs méthodes d'analyse. Nous avons ainsi pu montrer qu'un déplacement de charges transmembranaire photoinduit ultrarapide (<13 fs) apparaît dans la bactériorhodopsine. Le changement de moment dipolaire du rétinal (30 D) est augmenté d'un facteur proche de 1.5 par rapport au cas du rétinal en solution: il est donc favorisé par la présence du complexe protéique. La détection simultanée des réponses électronique et vibrationnelle ouvre des pistes sur la détermination du rôle fonctionnel de la polarisation initiale pour la dynamique structurale qui lui est subséquente.
• Observation of sub-100 ps conformational changes in photolyzed carbonmonoxy-myoglobin probed by time-resolved circular dichroism
  
  • Dartigalongue Thibault
  • Hache François
  , 2005. Conformational changes in proteins, which are known to play a paramount role in biophysical processes, are attracting much attention. For example, the change in carboxy-myoglobin (MbCO) after dissociation of the CO has recently been observed with a 100 ps time-resolution in a time-resolved X-Ray experiment. Shorter time resolution is however out of reach of such experiments. In order to investigate these processes on an ultrashort timescale, we have set up a time-resolved circular dichroism (CD) experiment in MbCO. The principle of the experiment is the following: after excitation with a pump beam, the CO-heme link breaks and a deoxy-heme structure appears very rapidly (< 1 ps). As the heme CD in the Soret region is very sensitive to the geometrical arrangement of the surrounding aromatic residues, measuring the change in the CD spectrum with time allows one to gain insight into the first steps of these conformational changes. The experiment is carried out on a 230 µM, pH 8.0 MbCO sample excited with a 400 nm pulse. The CD is measured across the Soret band as a function of time with a sub-picosecond resolution. After the initial drop in the CD due to the instantaneous electronic change of the heme, we observe a variation of the signal on a sub-100 picosecond timescale. In order to analyze these results, we have developed a calculation after Applequist's normal mode CD theory. Calculation of the contribution of the main residues to the rotational strength allows us to assign the observed signal to the stress provoked on the proximal histidine by the heme doming. This stress vanishes on a 100 ps timescale as the F-helix relaxes to its steady-state position. Extension of this experiment toward the far ultraviolet will provide a promising technique to investigate elementary changes in the secondary structure of proteins.
• Glu-Q-tRNAAsp synthetase coded by the yadB gene, a new paralog of aminoacyl-tRNA synthetase that glutamylates tRNAAsp anticodon
  
  • Blaise Mickaël
  • Becker Hubert F.
  • Lapointe Jacques
  • Cambillau Christian
  • Giegé Richard
  • Kern Daniel
  Biochimie, Elsevier, 2005, 87 (9-10), pp.847-861. Analysis of the completed genome sequences revealed presence in various bacteria of an open reading frame (ORF) encoding a polypeptide chain presenting important similarities with the catalytic domain of glutamyl-tRNA synthetases but deprived of the C-terminal anticodon-binding domain. This paralog of glutamyl-tRNA synthetases, the YadB protein, activates glutamate in the absence of tRNA and transfers the activated glutamate not on tRNA(Glu) but instead on tRNA(Asp). It has been shown that tRNA(Asp) is able to accept two amino acids: aspartate charged by aspartyl-tRNA synthetase and glutamate charged by YadB. The functional properties of YadB contrast with those of the canonical glutamyl-tRNA synthetases, which activate Glu only in presence of the cognate tRNA before aminoacylation of the 3'-end of tRNA. Biochemical approaches and mass spectrometry investigations revealed that YadB transfers the activated glutamate on the cyclopenthene-diol ring of the modified nucleoside queuosine posttranscriptionally inserted at the wobble position of the anticodon-loop to form glutamyl-queuosine. Unstability of the ester bond between the glutamate residue and the cyclopenthene-diol (half-life 7.5 min) explains why until now this modification escaped detection. Among Escherichia coli tRNAs containing queuosine in the wobble position, only tRNA(Asp) is substrate of YadB. Sequence comparison reveals a structural mimicry between the anticodon-stem and loop of tRNA(Asp) and the amino acid acceptor-stem of tRNA(Glu). YadB, renamed glutamyl-Q-tRNA(Asp) synthetase, constitutes the first enzyme structurally related to aminoacyl-tRNA synthetases which catalyzes a hypermodification in tRNA, and whose function seems to be conserved among prokaryotes. The discovery of glutamyl-Q-tRNA(Asp) synthetase breaks down the current paradigm according to which the catalytic domain of aminoacyl-tRNA synthetases recognizes the amino acid acceptor-stem of tRNA and aminoacylates the 3'-terminal ribose. The evolutionary significance of the existence of an aminoacyl-tRNA synthetase paralog dedicated to the hypermodification of a tRNA anticodon will be discussed. (10.1016/j.biochi.2005.03.007)
  DOI : 10.1016/j.biochi.2005.03.007
• Structure sensitivity in third-harmonic generation microscopy
  
  • Débarre Delphine
  • Supatto Willy
  • Beaurepaire Emmanuel
  Optics Letters, Optical Society of America - OSA Publishing, 2005, 30, pp.2134-2136. We characterize experimentally the influence of sample structure and beam focusing on signal level in third-harmonic generation (THG) microscopy. In the case of a homogeneous spherical sample, the dependence of the signal on the size of the sphere can be controlled by modifying the Rayleigh length of the excitation beam. More generally, the influence of excitation focusing on the signal depends on sample geometry, allowing one to highlight certain structures within a complex system. We illustrate this point by focusing-based contrast modulation in THG images of Drosophila embryos. (10.1364/OL.30.002134)
  DOI : 10.1364/OL.30.002134
• The CO oscillator as a probe of ligand dissociation dynamics in myoglobin
  
  • Ogilvie Jennifer P.
  • Polack Thomas
  • Franzen Stefan
  • Vos Marten H.
  • Joffre Manuel
  • Martin Jean-Louis
  • Alexandrou Antigoni
  , 2005, pp.ThD29. We report spectrally-integrated, visible-pump, mid-infrared probe studies of the CO ligand in myoglobin. Supported by density functional calculations, we find that the CO oscillator strength and frequency changes occur on disparate timescales following dissociation. © 2004 Optical Society of America
• Ultrafast conformational changes in carbonmonoxy-myoglobin after photolysis observed by time-resolved circular dichroism
  
  • Dartigalongue Thibault
  • Hache François
  , 2006, pp.583-587. An understanding of the first steps of conformational changes in proteins is very challenging. In that prospect, the processes occurring in carboxymyoglobin (MbCO) after ligand dissociation have been thoroughly studied. However, time-resolved absorption or Raman experiments, which probe localized electronic or vibrational modes of the protein, do not provide much information on the global conformation changes. Such is not the case for circular dichoism (CD), which is very sensitive to the geometry of the whole molecule. This chapter presents a subpicosecond time-resolved CD experiment on photolyzed MbCO and couples it with a CD calculation based on the polarizability theory to access information on the geometrical relaxation of the protein. Conformational changes are observed on a sub-100 ps timescale that is assigned to the strain brought onto the proximal histidine by the heme doming. (10.1016/B978-044452821-6/50085-X)
  DOI : 10.1016/B978-044452821-6/50085-X
• Terahertz polarimetry
  
  • Masson Jean-Baptiste
  • Gallot Guilhem
  , 2005, 3, pp.2111-2113. We present a simple and reliable technique to fully characterize the polarization state of a terahertz wave over a wide range of frequencies. Application to polarization ratio and crystal birefringence measurements is given. (10.1109/CLEO.2005.202385)
  DOI : 10.1109/CLEO.2005.202385
• Terahertz polarimetry
  
  • Masson Jean-Baptiste
  • Gallot Guilhem
  , 2005, pp.CFD2. We present a simple and reliable technique to fully characterize the polarization state of a terahertz wave over a wide range of frequencies. Application to polarization ratio and crystal birefringence measurements is given.
• Milieux de réaction pour l'activité thymidylate synthase de la ThyX
  
  • Myllykallio Hannu
  • Leduc Damien
  • Liebl Ursula
  , 2005. L'invention a pour but l'utilisation de milieux de réaction pour l'activité thymidylate synthase de la ThyX et leurs applications. En particulier, elle vise des milieux de réaction pour l'activité thymidylate synthase de la ThyX caractérisés en ce qu'ils comportent des flavines réduites et du CH2 H4 folate.
• Role of arginine 220 in the oxygen sensor FixL from Bradyrhizobium japonicum
  
  • Balland Véronique
  • Bouzhir-Sima Latifa
  • Kiger Laurent
  • Marden Michael C.
  • Vos Marten H.
  • Liebl Ursula
  • Mattioli Tony A.
  Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2005, 280, pp.15279-15288. In the heme-based oxygen sensor protein FixL, conformational changes induced by oxygen binding to the heme sensor domain regulate the activity of a neighboring histidine kinase, eventually restricting expression of specific genes to hypoxic conditions. The conserved arginine 220 residue is suggested to play a key role in the signal transduction mechanism. To obtain detailed insights into the role of this residue, we replaced Arg220 by histidine (R220H), glutamine (R220Q), glutamate (R220E), and isoleucine (R220I) in the heme domain FixLH from Bradyrhizobium japonicum. These mutations resulted in dramatic changes in the O2 affinity with Kd values in the order R220I < R220Q < wild type < R220H. For the R220H and R220Q mutants, residue 220 interacts with the bound O2 or CO ligands, as seen by resonance Raman spectroscopy. For the oxy-adducts, this H-bond modifies the π acidity of the O2 ligand, and its strength is correlated with the back-bonding-sensitive ν4 frequency, the koff value for O2 dissociation, and heme core-size conformational changes. This effect is especially strong for the wild-type protein where Arg220 is, in addition, positively charged. These observations strongly suggest that neither strong ligand fixation nor the displacement of residue 220 into the heme distal pocket are solely responsible for the reported heme conformational changes associated with kinase activity regulation, but that a significant decrease of the heme π* electron density because of strong back-bonding toward the oxygen ligand also plays a key role. (10.1074/jbc.M413928200)
  DOI : 10.1074/jbc.M413928200
• Observation of ultrafast conformational changes in carboxy-myoglobin by time-resolved circular dichroism
  
  • Dartigalongue Thibault
  • Hache François
  , 2005, 155 (2), pp.414-417. A time-resolved circular dichroism experiment is carried out on carboxy-myoglobin. CD is measured with a sub-picosecond time resolution after ligand dissociation. We observe a decrease of the CD signal in a few picoseconds followed by a 100 ps relaxation towards the deoxy-myoglobin values. Thanks to a calculation developed after the polarizability theory, we are able to assign this signal to a global reorganization of the protein conformation. (10.1016/j.synthmet.2005.09.026)
  DOI : 10.1016/j.synthmet.2005.09.026
• Observation of sub-100 ps conformational changes in photolyzed carbonmonoxy-myoglobin probed by time-resolved circular dichroism
  
  • Dartigalongue Thibault
  • Hache François
  , 2005.
• Multiphoton microscopy using intrinsic signals for pharmacological studies in unstained cardiac and vascular tissue
  
  • Beaurepaire Emmanuel
  • Boulesteix Thierry
  • Godeau G.
  • Pagès N.
  • Pena Ana-Maria
  • Sauviat Martin-Pierre
  • Schanne-Klein Marie-Claire
  • Senni K.
  , 2005, 5699, pp.67. We report two novel applications of multiphoton microscopy for pharmacological studies of unstained cardiovascular tissue. First, we show that second harmonic generation (SHG) microscopy of unstained cardiac myocytes can be used to determine the sarcomere length with sub-resolution accuracy, owing to the remarkable contrast of the SHG signal originating from myosin filaments. A measurement precision of 20 nm is achieved, taking the sample variability into account. We used this technique to measure sarcomere contracture in the presence of saxitoxin, and results were in agreement with mechanical measurements of atrial tissue contracture. Second, we characterized multiphoton microscopy of intact unlabeled arteries. We performed simultaneous detection of two-photon-excited fluorescence (2PEF) from elastin laminae and SHG from collagen fibers upon 860 nm excitation. Combined 2PEF/SHG images provide a highly specific, micron scale description of the architecture of these two major components of the vessel wall. We used this methodology to study the effects of lindane (a pesticide) on the artery wall structure and evidenced structural alteration of the vessel morphology. © (2005) COPYRIGHT SPIE-The International Society for Optical Engineering (10.1117/12.589558)
  DOI : 10.1117/12.589558
• In vivo analysis of Drosophila embryo developmental dynamics by femtosecond pulse-induced ablation and multimodal nonlinear microscopy
  
  • Supatto W.
  • Débarre Delphine
  • Moulia Bruno B.
  • Brouzés E.
  • Martin Jean-Louis
  • Schanne-Klein Marie-Claire
  • Farge Emmanuel
  • Beaurepaire Emmanuel
  , 2005, 5700, pp.256. Animal embryo development exhibits a complex ensemble of cell movements that are tightly regulated by developmental gene expression. It was proposed recently that mechanical factors may also play an important role during development. Investigating these dynamical processes is technically challenging and requires novel in vivo investigation methods. We show that multiphoton microscopy can be used for both perturbing and analyzing morphogenetic movements in vivo, (i) nonlinear microscopy is well adapted for the sustained imaging of early Drosophila embryos despite their highly scattering nature; (ii) femtosecond pulse-induced ablation can be used to process specific tissues in vivo. Combining this approach with multimodal microscopy (two-photon-excited fluorescence (2PEF) and third-harmonic generation (THG)), we report the successful quantitative modulation of morphogenetic movements in vivo. Our data provides insight to the issue of morphogenesis regulation. (10.1117/12.589425)
  DOI : 10.1117/12.589425
• Functionalized luminescent oxide nanoparticles for sodium channel imaging at the single molecule level
  
  • Casanova Didier
  • Giaume D.
  • Beaurepaire Emmanuel
  • Sauviat Martin-Pierre
  • Auksorius Egidijus
  • Buissette V.
  • Lahlil K.
  • Martin Jean-Louis
  • Gacoin Thierry
  • Boilot Jean-Pierre
  • Alexandrou Antigoni
  , 2005, 5705. Lanthanide-ion doped oxide nanoparticles were functionalized for use as fluorescent biological labels. These nanoparticles are synthesized directly in water which facilitates their functionalization, and are very photostable without emission intermittency. Nanoparticles functionalized with guanidinium groups act as artificial toxins and specifically target sodium channels. They are individually detectable in cardiac myocytes, revealing a heterogeneous distribution of sodium channels. Functionalized oxide nanoparticles appear as a novel tool particularly well adapted to long-term single-molecule tracking. © (2005) COPYRIGHT SPIE--The International Society for Optical Engineering (10.1117/12.590737)
  DOI : 10.1117/12.590737
• Observation of ultrafast conformational charge in carboxymyoglobin by time-resolved circular dichroism
  
  • Dartigalongue Thibault
  • Hache François
  , 2005.
• Spectroscopic analysis of keratin endogenous signal for skin multiphoton microscopy: erratum
  
  • Pena Ana-Maria
  • Strupler Mathias
  • Boulesteix Thierry
  • Godeau G.
  • Schanne-Klein Marie-Claire
  Optics Express, Optical Society of America - OSA Publishing, 2005, 13 (17), pp.6667. We present corrected versions of the list of authors and of Section 2.1. (10.1364/OPEX.13.006667)
  DOI : 10.1364/OPEX.13.006667
• Functionality of nitrated acetylcholine receptor: The two-step formation of nitrotyrosines reveals their differential role in effectors binding
  
  • Négrerie Michel
  • Martin Jean-Louis
  • Nghiêm H.-O.
  FEBS Letters, Wiley, 2005, 579 (12), pp.2643. The presence of nitrotyrosines is associated with several neurodegenerative pathologies. We evaluated the functionality of the nicotinic acetylcholine receptor possessing nitrotyrosines. The spectrum of the nitrated receptor displays an absorption band characteristic of ortho-nitrophenol. The presence of carbamylcholine in the agonist site prevented the effect of nitration by tetranitromethane in some conditions. The nitration occurred with two discrete steps and pointed out the differential involvement of tyrosines in the binding of acetylcholine and neurotoxin. We concluded that at least two residues involved in agonist binding can be nitrated, which bring similar contributions to the binding energy of the neurotransmitter. Cop. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. (10.1016/j.febslet.2005.03.084)
  DOI : 10.1016/j.febslet.2005.03.084
• Mid-infrared electric field characterization using a visible charge-coupled-device-based spectrometer
  
  • Kubarych Kevin J.
  • Joffre Manuel
  • Moore Amy
  • Belabas Nadia
  • Jonas David
  Optics Letters, Optical Society of America - OSA Publishing, 2005, 30 (10), pp.1228-1230. We characterize ultrashort mid-infrared pulses through upconversion by using the stretched pulses obtained from the uncompressed output of a chirped-pulse amplifier. The power spectrum thus translated into the visible region can be readily measured with a standard silicon CCD camera-based spectrometer. The spectral phase is also characterized by a variant of zero-added-phase spectral phase interferometry for direct electric field reconstruction. This is a general method that provides a multiplex advantage over conventional infrared detector array-based methods. © 2005 Optical Society of America (10.1364/OL.30.001228)
  DOI : 10.1364/OL.30.001228
• Activationless electron transfer through the hydrophobic core of cytochrome c oxidase
  
  • Jasaitis Audrius
  • Rappaport F.
  • Pilet Eric
  • Liebl Ursula
  • Vos Marten H.
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2005, 102 (31), pp.10882. Electron transfer (ET) within proteins occurs by means of chains of redox intermediates that favor directional and efficient electron delivery to an acceptor. Individual ET steps are energetically characterized by the electronic coupling V, driving force ΔG, and reorganization energy λ. λ reflects the nuclear rearrangement of the redox partners and their environment associated with the reactions; λ ≈ 700-1,100 meV (1 eV = 1.602 × 10-19 J) has been considered as a typical value for intraprotein ET. In nonphotosynthetic systems, functional ET is difficult to assess directly. However, using femtosecond flash photolysis of the CO-poised membrane protein cytochrome c oxidase, the intrinsic rate constant of the low-ΔG electron injection from heme a into the heme a 3-CuB active site was recently established at (1.4 ns)-1. Here, we determine the temperature dependence of both the rate constant and ΔG of this reaction and establish that this reaction is activationless. Using a quantum mechanical form of nonadiabatic ET theory and common assumptions for the coupled vibrational modes, we deduce that λ is <200 meV. It is demonstrated that the previously accepted value of 760 meV actually originates from the temperature dependence of CuB-CO bond breaking. We discuss that low-ΔG, low-λ reactions are common for efficiently channeling electrons through chains that are buried inside membrane proteins. (10.1073/pnas.0503001102)
  DOI : 10.1073/pnas.0503001102
• Etudes spectroscopiques du senseur à oxygène FixL
  
  • Jasaitis Audrius
  • Vos Marten H.
  • Balland Véronique
  • Mattioli T. A.
  • Bouzhir-Sima Latifa
  • Liebl Ursula
  , 2005, pp.poster.
• Sudden polarisation and coherent vibration in bacteriorhodopsin
  
  • Groma Geza
  • Colonna Anne
  • Joffre Manuel
  • Vos Marten H.
  • Martin Jean-Louis
  , 2005.