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

Articles

  • Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy.
    • Boulesteix Thierry
    • Beaurepaire Emmanuel
    • Sauviat Martin-Pierre
    • Schanne-Klein Marie-Claire
    Optics Letters, Optical Society of America - OSA Publishing , 2004, 29 (17), pp.2031-3 . We extend second-harmonic generation (SHG) microscopy to the measurement of sarcomere length in unstained living cardiac myocytes with 20-nm accuracy. We quantify individual sarcomere shortening in the presence of saxitoxin and find that it is in agreement with mechanical measurements of atrial tissue contracture. This functional application of SHG microscopy is generally applicable to quantify the physiological effects of drugs on contractile tissue. Our data also suggest that packed myosin heads in sarcomere thick filaments are responsible for the large second-harmonic endogenous signal in muscle tissue. (10.1364/OL.29.002031)
    DOI : 10.1364/OL.29.002031
  • From The Cover: An aminoacyl-tRNA synthetase-like protein encoded by the Escherichia coli yadB gene glutamylates specifically tRNAAsp
    • Dubois Daniel
    • Blaise Mickaël
    • Becker Hubert F.
    • Campanacci Valérie
    • Keith Gerard
    • Cambillau Christian
    • Lapointe Jacques
    • Kern Daniel
    • Becker D.
    • Giegé Richard
    Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2004, 101 (20), pp.7530-7535 . The product of the Escherichia coli yadB gene is homologous to the N-terminal part of bacterial glutamyl-tRNA synthetases (GluRSs), including the Rossmann fold with the acceptor-binding domain and the stem-contact fold. This GluRS-like protein, which lacks the anticodon-binding domain, does not use tRNA(Glu) as substrate in vitro nor in vivo, but aminoacylates tRNA(Asp) with glutamate. The yadB gene is expressed in wild-type E. coli as an operon with the dksA gene, which encodes a protein involved in the general stress response by means of its action at the translational level. The fate of the glutamylated tRNA(Asp) is not known, but its incapacity to bind elongation factor Tu suggests that it is not involved in ribosomal protein synthesis. Genes homologous to yadB are present only in bacteria, mostly in Proteobacteria. Sequence alignments and phylogenetic analyses show that the YadB proteins form a distinct monophyletic group related to the bacterial and organellar GluRSs (alpha-type GlxRSs superfamily) with ubiquitous function as suggested by the similar functional properties of the YadB homologue from Neisseria meningitidis. (10.1073/pnas.0401634101)
    DOI : 10.1073/pnas.0401634101
  • Application of the polarizability theory to the calculation of anisotropic circular dichroism spectra
    • Hache François
    • Dartigalongue Thibault
    Chemical Physics, Elsevier , 2004, 303 (1-2), pp.197-203 . We derive a general expression of the circular dichroism spectra of anisotropically distributed chiral molecules in the polarizability framework by introducing a tensorial rotational strength. Example calculation is carried out on an acridine-substituted Tröger base. It is shown that the circular dichroism is very sensitive to the direction propagation of the light beam. (10.1016/j.chemphys.2004.05.021)
    DOI : 10.1016/j.chemphys.2004.05.021
  • Resonant optical rectification in bacteriorhodopsin
    • Groma Géza
    • Colonna Anne
    • Lambry Jean-Christophe
    • Petrich Jacob
    • Váró G.
    • Joffre Manuel
    • Vos Marten H.
    • Martin Jean-Louis
    Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2004, 101 (21), pp.7971 . The relative role of retinal isomerization and microscopic polarization in the phototransduction process of bacteriorhodopsin is still an open question. It is known that both processes occur on an ultrafast time scale. The retinal trans?cis photoisomerization takes place on the time scale of a few hundred femtoseconds. On the other hand, it has been proposed that the primary light-induced event is a sudden polarization of the retinal environment, although there is no direct experimental evidence for femtosecond charge displacements, because photovoltaic techniques cannot be used to detect charge movements faster than picoseconds. Making use of the known high second-order susceptibility ?(2) of retinal in proteins, we have used a nonlinear technique, interferometric detection of coherent infrared emission, to study macroscopically oriented bacteriorhodopsin-containing purple membranes. We report and characterize impulsive macroscopic polarization of these films by optical rectification of an 11-fs visible light pulse in resonance with the optical transition. This finding provides direct evidence for charge separation as a precursor event for subsequent functional processes. A simple two-level model incorporating the resonant second-order optical properties of retinal, which are known to be a requirement for functioning of bacteriorhodopsin, is used to describe the observations. In addition to the electronic response, long-lived infrared emission at specific frequencies was observed, reflecting charge movements associated with vibrational motions. The simultaneous and phase-sensitive observation of both the electronic and vibrational signals opens the way to study the transduction of the initial polarization into structural dynamics. (10.1073/pnas.0306789101)
    DOI : 10.1073/pnas.0306789101
  • Functionalized fluorescent oxide nanoparticles: Artificial toxins for sodium channel targeting and imaging at the single-molecule level
    • Beaurepaire Emmanuel
    • Buissette Valérie
    • Sauviat Martin-Pierre
    • Giaume Domitile
    • Lahlil Khalid
    • Mercuri Antoine
    • Casanova Didier
    • Huignard Arnaud
    • Martin Jean-Louis
    • Gacoin Thierry
    • Boilot Jean-Pierre
    • Alexandrou Antigoni
    Nano Letters, American Chemical Society , 2004, 4 (11), pp.2079-2083 . 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 remarkably photostable without emission intermittency. Nanoparticles functiqnalized with guanidinium groups act as artificial toxins and specifically target sodium channels. They are individually detectable in live cardiac myocytes, revealing a heterogeneous distribution of sodium channels. Functionalized oxide nanoparticles appear to be a novel tool that is particularly attractive for long-term single-molecule tracking. (10.1021/nl049105g)
    DOI : 10.1021/nl049105g
  • Excited-state properties of flavin radicals in flavoproteins: Femtosecond spectroscopy of DNA photolyase, glucose oxidase, and flavodoxin
    • Pan Jie
    • Byrdin Martin
    • Aubert Corinne
    • Eker André P. M.
    • Brettel Klaus
    • Vos Marten H.
    Journal of Physical Chemistry B, American Chemical Society , 2004, 108 (28), pp.10160 . In isolated, catalytically inactive, DNA photolyase from Escherichia coli (E. coli), the flavin adenine dinucleotide cofactor is in its neutral radical state FAD?. It can be activated by a unique light-induced reduction of the flavin, a process initiated by the formation of the excited-state FAD?*. As the photophysical properties of this state are essentially unknown, we performed a comparative characterization by femtosecond transient absorption spectroscopy of FAD?* in DNA photolyase from E. coli and glucose oxidase from Aspergillus niger, and of the excited neutral radical flavin mononucleotide (FMN?*) in flavodoxin from Desulfovibrio gigas. In contrast to photolyase, in glucose oxidase and flavodoxin no electron-transfer products are observed after selective excitation of the flavin radical. In glucose oxidase, FAD?* decays to the ground state in 59 ± 5 ps, close to the 80-ps intrinsic lifetime of the excited state in photolyase, and we discuss that the intrinsic lifetime of the excited state of flavin radical in protein environment is in the 50-80 ps range. FMN?* in flavodoxin decays much faster (2.3 ± 0.3 ps), possibly because of quenching by formation of a very short-lived (INF 0.7 ps) electron-transfer intermediate. Spectroscopically, the excited state of FAD? in photolyase displays a pronounced spectral feature that is absent in the other systems studied. Further characterization by polarized photoselection experiments identifies the feature as an additional induced absorption band at ~ 550 nm superimposed on the ground-state bleaching signal. In view of the unique U-shape configuration of FAD in photolyase, we suggest it to reflect a flavin-adenine charge-transfer interaction.
  • CO Vibration as a Probe of Ligand Dissociation and Transfer in Myoglobin
    • Polack Thomas
    • Ogilvie Jennifer P.
    • Franzen S.
    • Vos Marten H.
    • Joffre Manuel
    • Martin Jean-Louis
    • Alexandrou Antigoni
    Physical Review Letters, American Physical Society , 2004, 93 (1), pp.18102 . We report femtosecond visible pump, midinfrared probe, spectrally integrated experiments resolving the dynamics of CO in myoglobin upon photodissociation. Our results show a progressive change in absorption strength of the CO vibrational transition during its transfer from the heme to the docking site, whereas the vibrational frequency change is faster than our time resolution. A phenomenological model gives good qualitative agreement with our data for a time constant of 400 fs for the change in oscillator strength. Density-functional calculations demonstrate that indeed vibrational frequency and absorption strength are not linearly coupled and that the absorption strength varies in a slower manner due to charge transfer from the heme iron to CO (10.1103/PhysRevLett.93.018102)
    DOI : 10.1103/PhysRevLett.93.018102
  • Intraprotein electron transfer and proton dynamics during photoactivation of DNA photolyase from E. coli: Review and new insights from an "inverse" deuterium isotope effect.
    • Byrdin Martin
    • Sartor Valérie
    • Eker André P.M.
    • Vos Marten H.
    • Aubert Corinne
    • Brettel Klaus
    • Mathis Paul
    Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier , 2004, 1655 (12 april), pp.64 . We review our work on electron transfer and proton dynamics during photoactivation in DNA photolyase from E. coli and discuss a recent theoretical study on this issue. In addition, we present unpublished data on the charge recombination between the fully reduced FADH- and the neutral (deprotonated) radical of the solvent exposed tryptophan W306. We found a pronounced acceleration with decreasing pH and an inverse deuterium isotope effect (kH/kD=0.35 at pL 6.5) and interpret it in a model of a fast protonation equilibrium for the W306 radical. Due to this fast equilibrium, two parallel recombination channels contribute differently at different pH values: one where reprotonation of the W306 radical is followed by electron transfer from FADH- (electron transfer time constant tet in the order of 10-50 µs), and one where electron transfer from FADH- (tet=25 ms) is followed by reprotonation of the W306 anion. Cop. 2004 Elsevier B.V. All rights reserved. (10.1016/j.bbabio.2003.07.001)
    DOI : 10.1016/j.bbabio.2003.07.001
  • NO binding and dynamics in reduced heme-copper oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus
    • Pilet Eric
    • Nitschke W.
    • Rappaport F.
    • Soulimane T.
    • Lambry Jean-Christophe
    • Liebl Ursula
    • Vos Marten H.
    Biochemistry, American Chemical Society , 2004, 43 (44), pp.14118 . Cytochrome c oxidase (CcO) has a high affinity for nitric oxide (NO), a property involved in the regulation of respiration. It has been shown that the recombination kinetics of photolyzed NO with reduced CcO from Paracoccus denitrificans on the picosecond time scale depend strongly on the NO/ enzyme stoichiometry and inferred that more than one NO can be accommodated by the active site, already at mildly suprastoichiometric NO concentrations. We have largely extended these studies by monitoring rebinding dynamics from the picosecond to the microsecond time scale, by performing parallel steady-state low-temperature electron paramagnetic resonance (EPR) characterizations on samples prepared similarly as for the optical experiments and comparing them with molecular-modeling results. A comparative study was performed on CcO ba3 from Thermus thermophilus, where two NO molecules cannot be copresent in the active site in the steady state because of its NO reductase activity. The kinetic results allow discrimination between different models of NO-dependent recombination and show that the overall NO escape probability out of the protein is high when only one NO is bound to CcO aa3, whereas strong rebinding on the 15-ns time scale was observed for CcO ba3. The EPR characterizations show similar results for aa3 at substoichiometric NO/enzyme ratios and for ba3, indicating formation of a 6-coordinate heme-NO complex. The presence of a second NO molecule in the aa3 active site strongly modifies the heme-NO EPR spectrum and can be rationalized by a rotation of the Fe-N-O plane with respect to the histidine that coordinates the heme iron. This proposal is supported by molecular-modeling studies that indicate a ~63° rotation of heme-bound NO upon binding of a second NO to the close-lying copper center CuB. It is argued that the second NO binds to CuB. (10.1021/bi0488808)
    DOI : 10.1021/bi0488808
  • Electron transfer between hemes in mammalian cytochrome c oxidase
    • Pilet Eric
    • Jasaitis Audrius
    • Liebl Ursula
    • Vos Marten H.
    Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2004, 101 (46), pp.16198 . Fast intraprotein electron transfer reactions associated with enzymatic catalysis are often difficult to synchronize and therefore to monitor directly in non-light-driven systems. However, in the mitochondrial respiratory enzyme cytochrome oxidase aa(3), the kinetics of the final electron transfer step into the active site can be determined: reverse electron flow between the close-lying and chemically identical hemes a(3) and a can be initiated by flash photolysis of CO from reduced heme a(3) under conditions where heme a is initially oxidized. To follow this reaction, we used transient absorption spectroscopy, with femtosecond time resolution and a time window extending to 4 ns. Comparison of the picosecond heme a(3)-CO photodissociation spectra under different redox states of heme a shows significant spectral interaction between both hemes, a phenomenon complicating the interpretation of spectral studies with low time resolution. Most importantly, we show that the intrinsic electron equilibration, corresponding to a DeltaG(0) of 45-55 meV, occurs in 1.2 +/- 0.1 ns. This is 3 orders of magnitude faster than the previously established equilibration phase of approximate to3 mus, which we suggest to reflect a change in redox equilibrium closely following CO migration out of the active site. Our results allow testing a number of conflicting predictions regarding this reaction between both experimental and theoretical studies. We discuss the potential physiological relevance of fast equilibration associated with this low-driving-force redox reaction. (10.1073/pnas.0405032101)
    DOI : 10.1073/pnas.0405032101
  • Coherent vibrational climbing in carboxyhemoglobin
    • Ventalon Cathie
    • Fraser James M.
    • Vos Marten H.
    • Alexandrou Antigoni
    • Martin Jean-Louis
    • Joffre Manuel
    Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2004, 101 (36), pp.13216-13220 . We demonstrate vibrational climbing in the CO stretch of carboxyhemoglobin pumped by midinfrared chirped ultrashort pulses. By use of spectrally resolved pump-probe measurements, we directly observed the induced absorption lines caused by excited vibrational populations up to v = 6. In some cases, we also observed stimulated emission, providing direct evidence of vibrational population inversion. This study provides important spectroscopic parameters on the CO stretch in the strong-field regime, such as transition frequencies and dephasing times up to the v = 6to v = 7 vibrational transition. We measured equally spaced vibrational transitions, in agreement with the energy levels of a Morse potential up to v = 6. It is interesting that the integral of the differential absorption spectra was observed to deviate far from zero, in contrast to what one would expect from a simple one-dimensional Morse model assuming a linear dependence of dipole moment with bond length. (10.1073/pnas.0401844101)
    DOI : 10.1073/pnas.0401844101
  • Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1
    • Graziani Sébastien
    • Xia Y.
    • Gurnon Jr
    • van Etten J.
    • Leduc Damien
    • Skouloubris Stéphane
    • Myllykallio Hannu
    • Liebl Ursula
    Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2004, 279 (52), pp.54340 . Sequence analysis of the 330-kb double-stranded DNA genome of Paramecium bursaria chlorella virus-1 revealed an open reading frame A674R that encodes a protein with up to 53% amino acid identity to a recently discovered new class of thymidylate synthases, called ThyX. Unlike the traditional thymidylate synthase, ThyA, that uses methylenetetrahydrofolate (CH(2)H(4)folate) as both a source of the methylene group and the reductant, CH(2)H(4)folate only supplies the methylene group in ThyX-catalyzed reactions. Furthermore, ThyX only catalyzes thymidylate (dTMP) formation in the presence of reduced pyridine nucleotides and oxidized FAD. The distribution and transcription patterns of the a674r gene in Chlorella viruses were examined. The a674r gene was cloned, and the protein was expressed in Escherichia coli. Biochemical characterization of the P. bursaria chlorella virus-1 recombinant ThyX protein indicates that it is more efficient at converting dUMP to dTMP than previously studied ThyX enzymes, thus allowing more detailed mechanistic studies of the enzyme. The ThyX-dUMP complexes with bound FAD function as efficient NAD(P) H oxidases, indicating that dUMP binds to the enzyme prior to NAD(P) H. This oxidation activity is directly linked to FAD reduction. Our results indicate that ThyX-specific inhibitors can be designed that do not affect ThyA enzymes. Finally, a model is proposed for the early stages of ThyX catalysis. (10.1074/jbc.M409121200)
    DOI : 10.1074/jbc.M409121200
  • Libration induced stretching mode excitation for pump-probe spectroscopy in pure liquid water
    • Amir Wafa
    • Gallot Guilhem
    • Hache François
    The Journal of Chemical Physics, American Institute of Physics , 2004, 121 (16), pp.7908 . We developed an experimental approach to study pure liquid water in the infrared and avoid thermal effects. This technique is based on libration induced stretching excitation of water molecules. A direct correspondence between frequencies within the libration and OH stretching bands is demonstrated. Energy diffusion is studied in pure liquid water by measuring wave packet dynamics of OH stretching vibrator with infrared femtosecond spectroscopy. Wave packet dynamics reveals ultrafast energy dynamics and reflects 130 fs intermolecular energy transfer between water vibrators. Energy diffusion is almost two orders of magnitude faster than self diffusion in water. Cop. 2004 American Institute of Physics. (10.1063/1.1800952)
    DOI : 10.1063/1.1800952
  • Photodissociation of heme distal methionine in ferrous cytochrome c revealed by subpicosecond time-resolved resonance raman spectroscopy
    • Cianetti Simona
    • Négrerie Michel
    • Vos Marten H.
    • Martin Jean-Louis
    • Kruglik Sergei G.
    Journal of the American Chemical Society, American Chemical Society , 2004, 126 (43), pp.13932 . Cytochrome c (cyt c) is anelectron-transfer heme protein that also binds nitric oxide (NO). In resting cyt c, two endogenous ligands of the heme iron are histidine-18 (His) and methionine-80 (Met) side chains, and NO binding requires the cleavage of one of the axial bonds. Previous femtosecond transient absorption studies suggested the photolysis of either Fe-His or Fe-Met bonds. We aimed at unequivocally identifying the internal side chain that is photodissociated in ferrous cyt c and at monitoring heme structural dynamics, by means of time-resolved resonance Raman (TR3) spectroscopy with ~0.6 ps time resolution. The Fe-His stretching mode at 216 cm-1 has been observed in photoproduct TR3 spectra for the first time for a c-type heme. The same transient mode was observed for a model ferrous cyt c N-fragment (residues 1-56) ligated with two His in the resting state. Our TR3 data reveal that upon ferrous cyt c photoexcitation, (i) distal Met side chain is instantly released, producing a five-coordinated domed heme structure, (ii) proximal His side chain, coupled to the heme, exhibits distortion due to strain exerted by the protein, and (iii) alteration in heme-cysteine coupling takes place along with the relaxation of the protein-induced deformations of the heme macrocycle. Copyright 2004 American Chemical Society. (10.1021/ja046442i)
    DOI : 10.1021/ja046442i
  • Dynamic regulation of the inducible nitric-oxide synthase by NO - Comparison with the endothelial isoform
    • Gautier Clément
    • Négrerie Michel
    • Wang Zq
    • Lambry Jean-Christophe
    • Stuehr Dennis
    • Collin Fabrice
    • Martin Jean-Louis
    • Slama-Schwok Anny
    Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2004, 279 (6), pp.4358 . We studied by ultrafast time-resolved absorption spectroscopy the geminate recombination of NO to the oxygenase domain of the inducible NO synthase, iNOSoxy, and to mutated proteins at position Trp-457. This tryptophan interacts with the tetrahydrobiopterin cofactor BH4, and W457A/F mutations largely reduced the catalytic formation of NO. BH4 decreases the rate of NO rebinding to the ferric iNOSoxy compared with that measured in its absence. The pterin has a larger effect on W457A/F than on the WT protein by increasing NO release from the protein. Therefore, BH4 raises the energy barrier for NO recombination to the mutated proteins in contrast with our observations on eNOS (SlamaSchwok, A., Negrerie, M., Berka, V., Lambry, J: C., Tsai, A.-L., Vos, M., and Martin, J.-L. (2002) J. Biol. Chem. 277, 7581-7586). Thus, we show a differential effect of BH4 on NO release from eNOS and iNOS. Compared with the position of this residue in the BH4-repleted enzyme, simulations of the NO dissociation dynamics point out at a swing of Trp-457 toward the missing pterin in the absence of BH4. NO geminate-rebinding data show a more efficient NO release from eNOS than from iNOS once NO is formed. Consistently, NO produced by iNOS is regulated by its ferric nitrosyl complex in contrast with eNOS. We show that the small enhancement of the NO geminate recombination rate in W457A/F compared with that in the WT enzyme cannot explain the decrease of NO yield because of the mutation; the major effect of the mutation thus arises from an uncoupled catalysis (Wang, Z. Q., Wei, C. C., Ghosh, S., Meade, A. L., Hemann, C., Hille, R., and Stuehr, D. J. (2001) Biochemistry 40, 12819-12825). (10.1074/jbc.M305048200)
    DOI : 10.1074/jbc.M305048200
  • Laser spectroscopic visualization of hydrogen bond motions in liquid water
    • Bratos S.
    • Leicknam J.-C.
    • Pommeret S.
    • Gallot Guilhem
    Journal of Molecular Structure, Elsevier , 2004, 708 (1-3), pp.197-203 . Ultrafast pump-probe experiments are described permitting a visualization of molecular motions in diluted HDO/D2O solutions. The experiments were realized in the mid-infrared spectral region with a time resolution of 150 fs. They were interpreted by a careful theoretical analysis, based on the correlation function approach of statistical mechanics. Combining experiment and theory, stretching motions of the OH?O bonds as well as HDO rotations were 'filmed' in real time. It was found that molecular rotations are the principal agent of hydrogen bond breaking and making in water. Recent literatures covering the subject, including molecular dynamics simulations, are reviewed in detail. Cop. 2004 Elsevier B.V. All rights reserved. (10.1016/j.molstruc.2004.06.036)
    DOI : 10.1016/j.molstruc.2004.06.036
  • Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers
    • Leduc Damien
    • Graziani Sébastien
    • Lipowski Gérard
    • Marchand C.
    • Le Maréchal P.
    • Liebl Ursula
    • Myllykallio Hannu
    Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2004, 101 (19), pp.7252-7257 . Little is known about the catalytic mechanism of the recently discovered ThyX family of flavin-dependent thymidylate synthases that are required for thymidylate (deoxythymidine 5'-monophosphate) synthesis in a large number of microbial species. Using a combination of site-directed mutagenesis and biochemical measurements, we have identified several residues of the Helicobacter pylori ThyX protein with crucial roles in ThyX catalysis. By providing functional evidence that the active site(s) of homotetrameric ThyX proteins is formed by three different subunits, our findings suggest that ThyX proteins have evolved through multimerization of inactive monomers. Moreover, because the active-site configurations of ThyX proteins, present in many human pathogenic bacteria, and of human thymidylate synthase ThyA are different, our results will aid in the identification of compounds specifically inhibiting microbial growth. (10.1073/pnas.0401365101)
    DOI : 10.1073/pnas.0401365101
  • Dynamique ultrarapide du proton dans l'eau : étude expérimentale
    • Amir Wafa
    • Gallot Guilhem
    • Hache François
    Journal de Physique IV Proceedings, EDP Sciences , 2004, 119, pp.111-112 . Nous présentons une étude de la dynamique femtoseconde du proton dans l'eau liquide par spectroscopie vibrationnelle femtoseconde résolue en temps. C'est à ce jour la première observation expérimentale d'une forme limite du proton en solution, et une première étape vers la compréhension du mécanisme de Grotthuss. (10.1051/jp4:2004119015)
    DOI : 10.1051/jp4:2004119015
  • Spectroscopie d'émission infrarouge femtoseconde des protéines photoréceptrices orientées
    • Colonna Anne
    • Lambry Jean-Christophe
    • Groma G.I.
    • Martin Jean-Louis
    • Vos Marten H.
    Journal de Physique IV Proceedings, EDP Sciences , 2004, 119, pp.161-162 . Ce travail a pour but l'étude de la dynamique primaire des protéines et de leur rôle dans la fonction du complexe protéique. La technique utilisée est une spectroscopie femtoseconde non linéaire ( $\chi ^{(2)}$) dans le domaine moyen infrarouge. L'ensemble des mouvements de charge induits par une excitation ultra-brève (~11fs) d'un échantillon de membranes orientées de bactériorhodopsine, un analogue bactérien de la protéine photoréceptrice du système visuel chez les mammifères, donne lieu à une émission directionnelle infrarouge (redressement optique). Les caractéristiques de cette émission (fréquence, phase, amplitude, direction) reflètent la réponse électronique et vibrationnelle de la molécule. (10.1051/jp4:2004119037)
    DOI : 10.1051/jp4:2004119037
  • Spectroscopy of electronic states in InAs quantum dots grown on In(x)Al(1-x)As/InP(001)
    • Fossard F.
    • Helman A.
    • Fishman G.
    • H Julien F.
    • Brault J.
    • Gendry M.
    • Peronne Emmanuel
    • Alexandrou Antigoni
    • E Schacham S.
    • Bahir G.
    • Finkman E.
    Physical Review Letters, American Physical Society , 2004, 69, pp.155333 . We have investigated optical properties of high-density InAs self-assembled quantum dots (QDs) in an InxAl1-xAs matrix, lattice matched to an InP (001) substrate. The weak lattice mismatch (~3%) results in a 90% coverage of the InxAl1-xAs surface with InAs QDs. By means of interband and intraband spectroscopies crossed with atomic force microscopy (AFM) measurements, we have determined that the InAs QDs optical properties depend on the deposited amount of InAs. Photoinduced absorption spectroscopy has been used to investigate midinfrared intraband absorptions. For three monolayers (ML) InAs deposit thickness, just above two-dimensional (2D)/3D growth mode transition (2.5 ML), the islands form as isolated elliptical dots elongated along the [10] direction and exhibit intraband resonances polarized either along the [110] or the [10] direction. For thicker deposition (>3 ML), InAs islands form chains of elliptical dots along the [10] direction where the quantum confinement is lost, resulting in a quantum-wire-like behavior. In this paper, we also report on photoluminescence and photocurrent spectroscopies, in order to get insight into the InAs/InxAl1-xAs island band structure. These experimental results are in good agreement with that of a multiband k*p model. (10.1103/PhysRevB.69.155333)
    DOI : 10.1103/PhysRevB.69.155333
  • Picosecond Dynamics of a Peptide from the Acetylcholine Receptor Interacting with a Neurotoxin Probed by Tailored Tryptophan Fluorescence
    • Chowdhury P.
    • Gondry Muriel
    • Genet Roger
    • Martin Jean-Louis
    • Ménez André
    • Négrerie Michel
    • Petrich Jacob
    Photochemistry and Photobiology, Blackwell Wiley [1962-....] , 2003, 77 (2), pp.151 . A tryptophan analog, dehydro-N-acetyl-l-tryptophanamide (Δ-NATA), which is produced enzymatically vial-tryptophan 2′,3′-oxidase from Chromobacterium violaceum, is newly used for time-resolved fluorescence. The absorption and emission maxima of Δ-NATA at 332 and 417 nm, respectively, in 20% dimethylformamide-water are significantly shifted to the red with respect to those of tryptophan in water, permitting us to measure its fluorescence in the presence of tryptophan residues. We demonstrate that the steady-state spectra and the fluorescence decay of Δ-NATA are very sensitive to environment, changing dramatically with solvent as the chromophore is localized within a protein and when this tagged protein binds to a peptide. The tryptophan oxidase was also used to modify the single Trp of a neurotoxin from snake (Naja nigricollis) venom. Modification of the toxin α (dehydrotryptophan-toxin α) permitted its investigation in complex with a synthetic 15-amino acid peptide corresponding to a loop of the agonist-binding site of acetylcholine receptor (AchR) from Torpedo marmorata species. The peptide α-185 possesses a single Trp at the third position (Trp187 of AchR) and a disulfide bridge between Cys192 and Cys193. A single-exponential rotational diffusion time with a constant of 1.65 ns is measured for the isolated 15-amino acid peptide. This suggests that Trp motion in the peptide in solution is strongly correlated with the residues downstream the peptide sequence, which may in part be attributed to long-range order imposed by the disulfide bond. The dynamics of the bound peptide are very different: the presence of two correlation times indicates that the Trp187 of the peptide has a fast motion (τr1= 140 ps and r(0)1= 0.14) relative to the overall rotation of the complex (τr2= 3.4 ns and r(0)2= 0.04). The correlation of the Trp residue with its neighboring amino acid residues and with the overall motion of the peptide is lost, giving rise to its rapid restricted motion. Thus, the internal dynamics of interacting peptides change on binding. (10.1562/0031-8655(2003)0770151PDOAPF2.0.CO2)
    DOI : 10.1562/0031-8655(2003)0770151PDOAPF2.0.CO2
  • Dissection of the triple tryptophan electron transfer chain in Escherichial coli DNA photolyase: Trp382 is the primary donor in photoactivation
    • Byrdin Martin
    • Eker André
    • Vos Marten H.
    • Brettel Klaus
    Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2003, 100 (15), pp.8676 . In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH*) induces an electron transfer over ≈15 Å from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH* ← W382 ← W359 ← W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH* decayed with a time constant τ ≈ 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived (τ ≈ 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH* is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield (≈0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin/neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH* is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH* (τ ≈ 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH* in WT and from W359 to excited FADH* in W382F mutant photolyase were estimated and related to the donor-acceptor distances. (10.1073/pnas.1531645100)
    DOI : 10.1073/pnas.1531645100
  • Precise alignment of a longitudinal Pockels cell for time-resolved circular dichroism experiments
    • Dartigalongue Thibault
    • Hache François
    Journal of the Optical Society of America B, Optical Society of America , 2003, 20 (8), pp.1780 . Artifacts in time-resolved circular dichroism experiments are carefully analyzed. We show that alignment of the longitudinal Pockels cell that modulates the laser polarization in such experiments is crucial. By developing a calculation of the behavior of a slightly misaligned Pockels cell, we are able to propose a simple alignment procedure. This procedure is based on the use of an analyzer at 0° or 45° of the Pockels cell axes, and it allows us to improve the alignment by 3 orders of magnitude and to reduce the artifact below the noise level in our experimental setup. Cop. 2003 Optical Society of America. (10.1364/JOSAB.20.001780)
    DOI : 10.1364/JOSAB.20.001780
  • Time-domain interferometry for direct electric-field reconstruction of mid-infrared femtosecond pulses
    • Ventalon Cathie
    • Raser James-M.
    • Joffre Manuel
    Optics Letters, Optical Society of America - OSA Publishing , 2003, 28 (19), pp.1826-1828 . Mid-infrared ultrashort pulses of 9.2-mu;m center wavelength are characterized in both amplitude and phase. This is achieved by use of a variant of spectral phase interferometry for direct electric field reconstruction in which spectral interferometry has been replaced with time-domain interferometry, a technique that is well suited for infrared pulses. The setup permits simultaneous recording of the second-order interferometric autocorrelation, thus providing an independent check on the retrieved spectral phase. Mid-infrared ultrashort pulses of 9.2-mu;m center wavelength are characterized in both amplitude and phase. This is achieved by use of a variant of spectral phase interferometry for direct electric field reconstruction in which spectral interferometry has been replaced with time-domain interferometry, a technique that is well suited for infrared pulses. The setup permits simultaneous recording of the second-order interferometric autocorrelation, thus providing an independent check on the retrieved spectral phase. Cop. 2003 Optical Society of America (10.1364/OL.28.001826)
    DOI : 10.1364/OL.28.001826
  • Life without dihydrofolate reductase FolA
    • Myllykallio H.
    • Leduc D.
    • Filee J.
    • Liebl Ursula
    Trends in Microbiology, Elsevier , 2003, 11 (5), pp.220 . Reduced folate derivatives participate in numerous reactions of bacterial intermediary metabolism. Consequently, the well-characterized enzyme implicated in the formation of tetrahydrofolate - dihydrofolate reductase FolA - was considered to be essential for bacterial growth. However, comparative genomics has revealed several bacterial genome sequences that appear to lack the folA gene. Here, we provide in silico evidence indicating that folA-lacking bacteria use a recently discovered class of flavin-dependent thymidylate synthases for deoxythymidine-5'-monophosphate synthesis, and propose that many bacteria must contain uncharacterized sources for reduced folate molecules that are still waiting to be discovered. (10.1016/S0966-842X(03)00101-X)
    DOI : 10.1016/S0966-842X(03)00101-X