Articles

Measurement of the Second-Order Hyperpolarizability of the Collagen Triple Helix and Determination of Its Physical Origin
- Deniset-Besseau Ariane
- Duboisset Julien
- Benichou Emmanuel
- Hache François
- Brevet Pierre-Francois
- Schanne-Klein Marie-Claire
Journal of Physical Chemistry B, American Chemical Society , 2009, 113 (40), pp.13437-13445 . We performed Hyper-Rayleigh Scattering (HRS) experiments to measure the second-order nonlinear optical response of the collagen triple helix and determine the physical origin of second harmonic signals observed in collagenous tissues. HRS experiments yielded a second-order hyperpolarizability of 1.25 x 10(-27) esu for rat-tail type I collagen, a Surprisingly large value considering that collagen presents no strong harmonophore in its amino acid sequence. Polarization-resolved experiments showed intramolecular coherent contributions to the HRS signal along with incoherent contributions that are the only contributions for molecules with dimensions much smaller than the excitation wavelength. We therefore modeled the effective second-order hyperpolarizability of the 290 nm long collagen triple helix by summing coherently the nonlinear response of well-aligned moieties along the triple helix axis. This model was confirmed by HRS measurements after denaturation of the collagen triple helix and for a collagen-like short model peptide [(Pro-Pro-Gly)(10)](3). We concluded that the large collagen nonlinear response originates in the tight alignment of a large number of small and weakly efficient harmonophores, presumably the peptide bonds, resulting in a coherent amplification of the nonlinear signal. (10.1021/jp9046837)
DOI : 10.1021/jp9046837
Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells
- Casanova Didier
- Bouzigues Cédric
- Nguyên Thanh-Liêm
- Ramodiharilafy Rivo O.
- Bouzhir-Sima Latifa
- Gacoin Thierry
- Boilot Jean-Pierre
- Tharaux Pierre-Louis
- Alexandrou Antigoni
Nature Nanotechnology, Nature Publishing Group , 2009, 4 (9), pp.581 . Low concentrations of reactive oxygen species, notably hydrogen peroxide (H 2 O 2), mediate various signalling processes in the cell. Production of these signals is highly regulated and a suitable probe is needed to measure these events. Here, we show that a probe based on a single nanoparticle can quantitatively measure transient H 2 O 2 generation in living cells. The Y 0.6 Eu 0.4 VO 4 nanoparticles undergo photoreduction under laser irradiation but re-oxidize in the presence of oxidants, leading to a recovery in luminescence. Our probe can be regenerated and reliably detects intracellular H 2 O 2 with a 30-s temporal resolution and a dynamic range of 1-45?M. The differences in the timing of intracellular H 2 O 2 production triggered by different signals were also measured using these nanoparticles. Although the probe is not selective towards H 2 O 2, in many signalling processes H 2 O 2 is, however, the dominant oxidant. In conjunction with appropriate controls, this probe is a powerful tool for unravelling pathways that involve reactive oxygen species. Cop. 2009 Macmillan Publishers Limited. All rights reserved. (10.1038/nnano.2009.200)
DOI : 10.1038/nnano.2009.200
Multimodal Multiphoton Imaging of Human Eye Tissues
- Aptel Florent
- Olivier Nicolas
- Deniset Ariane
- Plamann Karsten
- Denis P.
- Legeais Jean-Marc
- Schanne-Klein Marie-Claire
- Beaurepaire Emmanuel
Investigative Ophthalmology & Visual Science, Association for Research in Vision and Ophthalmology , 2009, 50, pp.E-Abstract 3693 . Purpose:To evaluate three combined modalities of multiphoton microscopy, second-harmonic generation (SHG), third-harmonic generation (THG), and two-photon-excited fluorescence (2PEF) for imaging cornea and trabecular meshwork in human intact eye tissue. Methods:A tunable femtosecond laser chain ( = 700-1250 nm) comprising a titanium-sapphire laser oscillator and an optical parametric oscillator was used to produce 2PEF (380-620nm), SHG (/2=430 or 600nm) and THG (/3= 400nm). Eight corneoscleral discs from eye bank and seven fresh corneal buttons obtained after penetrating keratoplasty were examined with water-immersion objectives. Forward and backward signals were detected and compared. Results:The three imaging modalities provide complementary information on intact tissue over the entire thickness of the cornea. THG imaging reveals the tissue morphology, including the epithelium structure with sub-cellular resolution. Polarization-resolved THG microscopy reveals stromal birefringent domains. In phenol-stained corneas, THG also reveals the keratocytes network. SHG imaging probes the distribution of stromal collagen lamellas organization. 2PEF imaging reveals the elastic component of the extra-cellular matrix and the distribution of fluorescent organelles (i.e. mitochondria) in stromal and epithelial cells. The trabeculum images show the three-dimensional organisation of the trabecular lamellas. Emission is predominantly forward directed for THG and SHG but in some cases, images can be recorded in the epi-direction. Conclusions:The combined imaging modalities of SHG, THG, and 2PEF microscopy are effective methods to evaluate cornea and trabecular meshwork microstructures in situ. This imaging approach should prove particularly appropriate for assessing corneal and glaucoma physiopathology, and might be amenable to in vivo diagnostics.
Femtosecond spectroscopy from the perspective of a global multidimensional response function
- Nuernberger Patrick
- Lee Kevin F.
- Joffre Manuel
Accounts of Chemical Research, American Chemical Society , 2009, 42 (9), pp.1433-1441 . At the microscopic level, multidimensional response functions, such as the nonlinear optical susceptibility or the time-ordered response function, are commonly used tools in nonlinear optical spectroscopy for determining the nonlinear polarization resulting from an arbitrary excitation. In this Account, we point out that the approach successfully developed for the nonlinear polarization can also be used in the case of a directly observable macroscopic quantity. This observable can be, for example, the electric field radiated in a nonlinear mixing experiment, the rate of fluorescence resulting from one- or two-photon absorption, or the rate of a photochemical reaction. For each of these physical processes, perturbation theory can be used to expand the measured quantity in a power series of the exciting field, and an appropriate global response function can be introduced for each order of perturbation. At order n, the multidimensional response function will depend on n variables (either time or frequency) and have the same general properties as the nonlinear susceptibility resulting, for example, from time invariance or causality. The global response function is introduced in this Account in close analogy with the nonlinear susceptibility or the time-ordered microscopic response. We discuss various applications of the global response function formalism. For example, it can be shown that in the weak field limit, a stationary signal induced in a time-invariant system is independent of the spectral phase of the exciting field. Although this result had been demonstrated previously, the global response function enables its derivation in a more general way because no specific microscopic model is needed. Multidimensional spectroscopy is obviously ideally suited to measure the global multidimensional response function. It is shown that the second (or third)-order response can be exactly measured with 2D (or 3D) spectroscopy by taking into account the exact shape of the exciting pulses. In the case of a 2D measurement of the third-order response, a particular projection of the complete 3D response function is actually measured. This projection can be related to a mixed time and frequency representation of the response function when the pulses are assumed to be infinitely short. We thus show that the global response function is a useful tool for deriving general results and that it should help in designing future experimental schemes for femtosecond spectroscopy. Cop. 2009 American Chemical Society. (10.1021/ar900001w)
DOI : 10.1021/ar900001w
Application of time-resolved circular dichroism to the study of conformational changes in photochemical and photobiological processes
- Hache François
Journal of Photochemistry and Photobiology A: Chemistry, Elsevier , 2009, 204 (2-3), pp.137-143 . Circular dichroism is known to be a very sensitive probe of the molecular conformation and implementation of this technique in a pump-probe experiment is very appealing to access information on the dynamics of conformational changes occurring in photochemical or photobiological processes. In the past years, we have developed such techniques in various ways and applied them to several chemical or biological studies which are presented in this article. Applications concern spectroscopic studies of the excited state in ruthenium tris(bipyridyl) or tris(phenanthroline), dynamics of conformational changes in photoexcited binaphthol and study of the conformational changes occurring in photolyzed carboxy-myoglobin. Extension of these techniques towards biological issues such as protein folding is discussed. Cop. 2009 Elsevier B.V. All rights reserved. (10.1016/j.jphotochem.2009.03.012)
DOI : 10.1016/j.jphotochem.2009.03.012
Inferring maps of forces inside cell membrane microdomains
- Masson J.-B.
- Casanova Didier
- Türkcan Silvan
- Voisinne G.
- Popoff Michel
- Vergassola M.
- Alexandrou Antigoni
Physical Review Letters, American Physical Society , 2009, 102 (4), pp.48103 . Mapping of the forces on biomolecules in cell membranes has spurred the development of effective labels, e.g., organic fluorophores and nanoparticles, to track trajectories of single biomolecules. Standard methods use particular statistics, namely the mean square displacement, to analyze the underlying dynamics. Here, we introduce general inference methods to fully exploit information in the experimental trajectories, providing sharp estimates of the forces and the diffusion coefficients in membrane microdomains. Rapid and reliable convergence of the inference scheme is demonstrated on trajectories generated numerically. The method is then applied to infer forces and potentials acting on the receptor of the toxin labeled by lanthanide-ion nanoparticles. Our scheme is applicable to any labeled biomolecule and results show its general relevance for membrane compartmentation. Cop. 2009 The American Physical Society. (10.1103/PhysRevLett.102.048103)
DOI : 10.1103/PhysRevLett.102.048103
Apports récents des techniques de quantification de la fibrose pour l'examen anatomopathologique en transplantation rénale
- Servais A.
- Meas-Yedid V.
- Morelon E.
- Strupler Mathias
- Schanne-Klein Marie-Claire
- Legendre C.
- Olivo-Marin J.-C.
- Thervet É.
Médecine/Sciences, EDP Sciences , 2009, 25 (11), pp.945-950 . La néphropathie chronique d'allogreffe constitue la cause principale de perte des greffons rénaux à long terme. Elle peut être détectée précocement par des biopsies de dépistage effectuées de manière systématique. La classification usuelle, semi-quantitative, souffre d'une mauvaise reproductibilité. Diverses méthodes morphométriques ont donc été développées pour quantifier la fibrose interstitielle qui caractérise cette néphropathie. Certaines utilisent la coloration spécifique par le rouge Sirius. L'analyse d'image couleur par segmentation permet une quantification automatique, rapide et robuste de la fibrose interstitielle. Elle utilise une segmentation couleur associée à une analyse de couleur, de localisation spatiale et de forme sur des biopsies colorées au trichrome de Masson. À l'avenir, l'étude des collagènes fibrillaires par la génération de second harmonique pourrait permettre une approche spécifique des composants de la fibrose. (10.1051/medsci/20092511945)
DOI : 10.1051/medsci/20092511945
Unobtrusive interferometer tracking by path length oscillation for multidimensional spectroscopy
- Lee Kevin F.
- Bonvalet Adeline
- Nuernberger Patrick
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2009, 17 (15), pp.12379-12384 . We track the path difference between interferometer arms with few-nanometer accuracy without adding optics to the beam path. We measure the interference of a helium-neon beam that copropagates through the interferometer with midinfrared pulses used for multidimensional spectroscopy. This can indicate motion, but not direction. By oscillating the path length of one arm with a mirror on a piezoelectric stack and monitoring the oscillations of the recombined helium-neon beam, the direction can be calculated, and the path delay can be continuously tracked. © 2009 Optical Society of America. (10.1364/OE.17.012379)
DOI : 10.1364/OE.17.012379
Suppression of perturbed free-induction decay and noise in experimental ultrafast pump-probe data
- Nuernberger Patrick
- Lee Kevin F.
- Bonvalet Adeline
- Polack Thomas
- Vos Marten H.
- Alexandrou Antigoni
- Joffre Manuel
Optics Letters, Optical Society of America - OSA Publishing , 2009, 34 (20), pp.3226-3228 . We apply a Fourier filtering technique for the global removal of coherent contributions, like perturbed freeinduction decay, and noise, to experimental pump-probe spectra. A further filtering scheme gains access to spectra otherwise only recordable by scanning the probe's center frequency with adjustable spectral resolution. These methods cleanse pump-probe data and allow improved visualization and simpler analysis of the contained dynamics. We demonstrate these filters using visible pump/mid-infrared probe spectroscopy of ligand dissociation in carboxyhemoglobin. Cop. 2009 Optical Society of America. (10.1364/OL.34.003226)
DOI : 10.1364/OL.34.003226
Heme ligand binding properties and intradimer interactions in the full-length sensor protein Dos from Escherichia coli and its isolated heme domain
- Lechauve C.
- Bouzhir-Sima Latifa
- Yamashita Taku
- Marden M.C.
- Vos Marten H.
- Liebl Ursula
- Kiger L.
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2009, 284 (52), pp.36146 . Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant ∼1 μm), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O2) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated ∼10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins. (10.1074/jbc.M109.066811)
DOI : 10.1074/jbc.M109.066811
Extended fano model of extraordinary electromagnetic transmission through subwavelength hole arrays in the terahertz domain
- Masson Jean-Baptiste
- Podzorov Alexander
- Gallot Guilhem
Optics Express, Optical Society of America - OSA Publishing , 2009, 17 (17), pp.15280-15291 . We developed an extended Fano model describing the Extraordinary Electromagnetic Transmission (EET) through arrays of subwavelength apertures, based on terahertz transmission measurements of arrays of various hole size and shapes. Considering a frequency-dependent coupling between resonant and non-resonant pathways, this model gives access to a simple analytical description of EET, provides good agreement with experimental data, and offers new parameters describing the influence of the hole size and shape on the transmitted signal. Cop. 2009 Optical Society of America. (10.1364/OE.17.015280)
DOI : 10.1364/OE.17.015280
Removing cross-phase modulation from midinfrared chirped-pulse upconversion spectra
- Lee Kevin F.
- Nuernberger Patrick
- Bonvalet Adeline
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2009, 17 (21), pp.18738-18744 . We observe that narrow spectral features in mid-infrared spectra obtained by chirped-pulse up-conversion are strongly distorted by crossphase modulation between the mid-infrared field and the chirped pulse. We discuss the consequences of this effect on spectral resolution, and introduce a correction method that recovers masked lines. This simple correction can be applied either when the upconverted field is fully characterized, such as in multidimensional spectroscopy, or when causality can be used, such as in absorption spectroscopy, which we demonstrate experimentally. Cop.2009 Optical Society of America. (10.1364/OE.17.018738)
DOI : 10.1364/OE.17.018738
HIV-1 IN alternative molecular recognition of DNA induced by raltegravir resistance mutations
- Mouscadet J.-F.
- Arora Rakesh
- André J.
- Lambry Jean-Christophe
- Delelis O.
- Malet I.
- Marcelin A.-G.
- Calvez V.
- Tchertanov L.
Journal of Molecular Recognition, Wiley , 2009, 22 (6), pp.480-494 . Virologic failure during treatment with raltegravir, the first effective drug targeting HIV integrase, is associated with two exclusive pathways involving either Q148H/R/K, G140S/A or N155H mutations. We carried out a detailed analysis of the molecular and structural effects of these mutations. We observed no topological change in the integrase core domain, with conservation of a newly identified V-shaped hairpin containing the Q148 residue, in particular. In contrast, the mutations greatly altered the specificity of DNA recognition by integrase. The native residues displayed a clear preference for adenine, whereas the mutant residues strongly favored pyrimidines. Raltegravir may bind to N155 and/or Q148 residues as an adenine bioisoster. This may account for the selected mutations impairing raltegravir binding while allowing alternative DNA recognition by integrase. This study opens up new opportunities for the design of integrase inhibitors active against raltegravir-resistant viruses. Copyright Cop. 2009 John Wiley and Sons, Ltd. Supporting information may be found in the online version of this article. (10.1002/jmr.970)
DOI : 10.1002/jmr.970
A model for thermal exchange in axons during action potential propagation.
- Masson Jean-Baptiste
- Gallot Guilhem
European Biophysics Journal, Springer Verlag (Germany) , 2008, 37 (6), pp.1001-1006 . Several experiments have shown that during propagation of the action potential in axons, thermal energy is locally exchanged. In this paper, we use a simple model based on statistical physics to show that an important part of this exchange comes from the physics of the effusion. We evaluate, during the action potential propagation, the variation of internal energy and of the energy associated with the chemical potential of the effusion of water and ions to extract the thermal energy exchanged. The temperature exchanged is then evaluated on the area where the action potential is active. Results give a good correspondence between experimental work and this model, showing that an important part of the thermal energy exchange comes from the statistical cooling power of the effusion. (10.1007/s00249-008-0329-5)
DOI : 10.1007/s00249-008-0329-5
Ultrafast heme-residue bond formation in six-coordinate heme proteins: implications for functional ligand exchange.
- Vos Marten H.
- Battistoni Andrea
- Lechauve Christophe
- Marden Michael C.
- Kiger Laurent
- Desbois Alain
- Pilet Eric
- de Rosny Eve
- Liebl Ursula
Biochemistry, American Chemical Society , 2008, 47 (21), pp.5718-23 . A survey is presented of picosecond kinetics of heme-residue bond formation after photolysis of histidine, methionine, or cysteine, in a broad range of ferrous six-coordinate heme proteins. These include human neuroglobin, a bacterial heme-binding superoxide dismutase (SOD), plant cytochrome b 559, the insect nuclear receptor E75, horse heart cytochrome c and the heme domain of the bacterial sensor protein Dos. We demonstrate that the fastest and dominant phase of binding of amino acid residues to domed heme invariably takes place with a time constant in the narrow range of 5-7 ps. Remarkably, this is also the case in the heme-binding SOD, where the heme is solvent-exposed. We reason that this fast phase corresponds to barrierless formation of the heme-residue bond from a configuration close to the bound state. Only in proteins where functional ligand exchange occurs, additional slower rebinding takes place on the time scale of tens of picoseconds after residue dissociation. We propose that the presence of these slower phases reflects flexibility in the heme environment that allows external ligands (O2, CO, NO, . . .) to functionally replace the internal residue after thermal dissociation of the heme-residue bond. (10.1021/bi800288z)
DOI : 10.1021/bi800288z
Collective behavior during the exit of a wetting liquid through a network of channels
- Baroud Charles N.
- Wang Xin C.
- Masson Jean-Baptiste
Journal of Colloid and Interface Science, Elsevier , 2008, 326 (2), pp.445 . The exit of a wetting fluid from a thin microchannel into a sudden expansion is studied experimentally. In the case of the exit from a single channel, the advancing interface converges to a parabolic shape after an initial transient, in accordance with the lubrication limit analysis of a spreading drop. The experiments are then repeated for the exit from two parallel channels. At early times, the two exiting drops behave independently and display the same evolution as a single exiting droplet, while at late times we recover a single parabolic profile. The transition between the early and late states is due to the merging of the two drops, which is associated with a sudden increase in the flow rate. This is the signature of a collective effect which acts to redistribute the fluid spatially. Finally, the experiment is generalized to the case of seven parallel channels where a cascade of two-by-two mergings is observed, indicating that local interactions dominate the dynamics which lead to the global state of the system. Cop. 2008 Elsevier Inc. All rights reserved. (10.1016/j.jcis.2008.06.040)
DOI : 10.1016/j.jcis.2008.06.040
Second Harmonic Microscopy to Quantify Renal Interstitial Fibrosis and Arterial Remodeling
- Strupler Mathias
- Hernest Monica
- Fligny Cécile
- Martin Jean-Louis
- Tharaux Pierre-Louis
- Schanne-Klein Marie-Claire
Journal of Biomedical Optics, Society of Photo-optical Instrumentation Engineers , 2008, 13 (5), pp.054041 . Interstitial ﬁbrosis is a powerful pejorative predictor of progression of nephropathies in a variety of chronic renal diseases. It is characterized by the depletion of kidney cells and their replacement by extracellular matrix, in particular, type-I ﬁbrillar collagen, a protein scarce in normal interstitium. However, assessment of ﬁbrosis remains a challenge in research and clinical pathology. We develop a novel methodology based on second harmonic generation SHG microscopy, and we image collagen fibers in human and mouse unstained kidneys. We take into account the variability in renal shape, and we develop automated image processing for quantitative scoring of thick murine tissues. This approach allows quantitative 3-D imaging of interstitial ﬁbrosis and arterial remodeling with high accuracy. Moreover, SHG microscopy helps to raise pathophysiological questions. First, imaging of a large volume within a mouse kidney shows that progression of ﬁbrosis is a heterogeneous process throughout the different renal compartments. Second, SHG from ﬁbrillar collagens does not overlap with the glomerular tuft, despite patent clinical and experimental glomerulosclerosis. Since glomerulosclerosis involves SHG-silent nonﬁbrillar collagens, our work supports pathophysiological differences between interstitial ﬁbrosis and glomerulosclerosis, a clearly nonﬁbrotic process. © 2008 Society of Photo-Optical Instrumentation Engineers (10.1117/1.2981830)
DOI : 10.1117/1.2981830
Mechanical factors activate beta-catenin-dependent oncogene expression in APC1638N/+ mouse colon
- Whitehead J.
- Vignjevic Danijela
- Fütterer C.
- Beaurepaire Emmanuel
- Robine Sylvie
- Farge Emmanuel
HFSP Journal , 2008, 2 (5), pp.286 . catenin acts as a critical regulator of gastrointestinal homeostasis through its control of the Wnt signaling pathway, and genetic or epigenetic lesions which activate Wnt signaling are the primary feature of colon cancer. -catenin is also a key element of mechanotranscription pathways, leading to upregulation of master developmental gene expression during Drosophila gastrulation, or regulating mammalian bone development and maintenance. Here we investigate the impact of mechanical stimulation on the initiation of colon cancer. Myc and Twist1, two oncogenes regulated through -catenin, are expressed in response to transient compression in APC deﬁcient "APC1638N/+... colon tissue explants, but not in wild-type colon explants. Mechanical stimulation of APC1638N/+ tissue leads to the phosphorylation of -catenin at tyrosine 654, the site of interaction with E-cadherin, as well as to increased nuclear localization of -catenin. The mechanical activation of Myc and Twist1 expression in APC1638N/+ colon can be prevented by blocking -catenin phosphorylation using Src kinase inhibitors. Microenvironmental signals are known to cooperate with genetic lesions to promote the nuclear -catenin accumulation which drives colon cancer. Here we demonstrate that when APC is limiting, mechanical strain, such as that associated with intestinal transit or tumor growth, can be interpreted by cells of preneoplastic colon tissue as a signal to initiate a -catenin dependent transcriptional program characteristic of cancer (10.2976/1.2955566)
DOI : 10.2976/1.2955566
Processus ultra-rapides dans les hémoprotéines [Ultrafast processes in heme proteins]
- Vos Marten H.
L'Actualité Chimique, Société chimique de France (SCF) , 2008, 317 (52), pp.55 . Les hémoprotéines sont impliquées dans une grande diversité de fonctions biologiques qui incluent transport et stockage d'oxygène, catalyse, transfert d'électron et signalisation. Le fer de l'hème peut être lié aux acides aminés et également aux molécules diatomiques (O2, NO, CO). Ces liaisons peuvent être rompues par une impulsion lumineuse avec un rendement élevé. En utilisant des techniques de spectroscopie optique ultrarapide, cette propriété donne la possibilité unique d'étudier la dynamique structurelle et électronique de l'hème et de son environnement protéique à l'échelle femtoseconde-picoseconde, c'est-à-dire à l'échelle même des vibrations internes de la macromolécule. Cet article décrit des développements récents dans ce domaine, en particulier l'implication de mouvements concertés hème-protéine dans des réactions balistiques de transfert de ligands et la possibilité de suivre des étapes intermédiaires de propagation d'un " signal " au sein de protéines de signalisation. Heme proteins contribute to a large variety of biological functions including transport and storage of oxygen, catalysis, electron transfer and signalling. The iron atom in the heme can bind amino acids and also diatomic molecules (O-2, NO, CO). These bonds can efficiently be broken by a light pulse. Using ultrafast optical spectroscopy techniques, this property allows the study of structural and electronic dynamics in the heme and its proteic environment at femtosecond-picosecond time scales, i.e. at the time scale of internal vibrations of the macromolecular systems. This review describes recent progress in this domain, specifically the role of concerted movements of the heme-protein system in ballistic ligand transfer reactions as well as the real-time monitoring of signal propagation within signalling proteins.
Two photon-induced electron injection from a nanotrigger in native endothelial NO-synthase
- Beaumont Edward
- Lambry Jean-Christophe
- Robin A.-C.
- Martasek P.
- Blanchard-Desce M.
- Slama-Schwok Anny
ChemPhysChem, Wiley-VCH Verlag , 2008, 9 (16), pp.2325 . We have recently designed a nanotrigger (NT), a photoactive molecule addressing the NADPH sites of proteins. This nanotrigger has a 103 times larger two-photon cross-section compared to the ubiquitous NADPH cofactor. In this work, we tested whether two-photon excitation of the bound NT to NADPH sites may be used to initiate enzymatic catalysis by appropriate electron injection. To establish proof of principle, we monitored the ultrafast absorption of NT bound to the fully active endothelial NO-Synthase (eNOS) following excitation by one and two-photons at 405 and 810 nm, respectively. Electron injection from NT* to FAD in eNOS initiated the catalytic cycle in 15±3 ps at both exciting wavelengths. The data proved for the first time that electron transfer can be promoted by two-photon excitation. We also show that the nanotrigger decays faster in homogeneous solvents than in the NADPH site of proteins, suggesting that hindered environments modified the natural decay of NT. The nanotrigger provides a convenient way of synchronizing an ensemble of proteins in solution with a femtosecond laser pulse. The ability of NT to initiate NOS catalysis by two-photon excitation may be exploited for controlled and localized release of free NO in cells with enhanced spatial and temporal resolution. Cop. 2008 Wiley-VCH Verlag GmbH & Co. KGaA. (10.1002/cphc.200800411)
DOI : 10.1002/cphc.200800411
Fast ligand and electron transfer dynamics in oxidases and cytochrome c
- Vos Marten H.
Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics, Elsevier , 2008, 1777 (Supplement), pp.S66 . The active site of heme-copper oxidases contains two cofactors, heme a3 and CuB, which can both bind external ligands as the substrate O2 and signalling molecules NO and CO, and which are both involved in electron transfer processes. Over the last few years we have exploited the fact that the heme-ligand bond can be dissociated by a short light pulse to explore the dynamics of CO and NO in the active site and the interaction between the two cofactors using ultrafast spectroscopic techniques. For example, we have time-resolved the CO transfer from heme a3 to CuB and shown that it occurs in a ballistic way in ∼ 500 fs, which presumably reflects rigidity of the active site. Heme a is located close to heme a3 (∼ 7 Å edge-to-edge) and acts as electron donor for the active site. Using mixed valence oxidases we have extended the 'reverse electron flow' technique to the ultrafast regime and demonstrated that this electron transfer process occurs in only 1.2 ns. The process is activationless and associated with a very low reorganization energy (< 200 meV), in contrast to common assumptions but in general agreement with the hydrophobic environment of the reactants. Finally, ligand dynamics in native and modified cytochrome c reflects the rigidity required for optimal electron transfer properties. (10.1016/j.bbabio.2008.05.259)
DOI : 10.1016/j.bbabio.2008.05.259
Ultrafast dynamics of ligands within heme proteins
- Vos Marten H.
Biochimica et Biophysica Acta (BBA) - Reviews on Bioenergetics, Elsevier , 2008, 1777, pp.15-31 . Physiological bond formation and bond breaking events between proteins and ligands and their immediate consequences are difficult to synchronize and study in general. However, diatomic ligands can be photodissociated from heme, and thus in heme proteins ligand release and rebinding dynamics and trajectories have been studied on timescales of the internal vibrations of the protein that drive many biochemical reactions, and longer. The rapidly expanding number of characterized heme proteins involved in a large variety of functions allows comparative dynamics-structure-function studies. In this review, an overview is given of recent progress in this field, and in particular on initial sensing processes in signaling proteins, and on ligand and electron transfer dynamics in oxidases and cytochromes. (10.1016/j.bbabio.2007.10.004)
DOI : 10.1016/j.bbabio.2007.10.004
Conformational changes in photoexcited (R)-(+)-1,1'-bi-2-naphthol studied by time-resolved circular dichroism
- Niezborala Claire
- Hache François
Journal of the American Chemical Society, American Chemical Society , 2008, 130 (38), pp.12783-12786 . Conformational changes following photoexcitation of (R)-(+)-1,1′-bi-2-naphthol are studied with a time-resolved circular dichroism (CD) experiment. Two wavelengths are investigated. For λ = 237 nm, we observe a bleaching of the ground-state absorption and a transient CD structure. Thanks to a coupled-oscillator calculation, we can attribute this effect to a decrease of the dihedral angle. For λ = 245 nm, excited-state absorption and CD are observed. All these effects are solvent-dependent. In particular, it is shown that dynamics is slower in a protic solvent, which is attributed to hydrogen-bonding of the hydroxy groups with the solvent. (10.1021/ja8039844)
DOI : 10.1021/ja8039844
Nanofluidics in the Debye Layer at Hydrophilic and Hydrophobic Surfaces
- Bouzigues Cédric
- Tabeling Paul
- Bocquet L.
Physical Review Letters, American Physical Society , 2008, 101, pp.114503 . By using evanescent waves, we study equilibrium and dynamical properties of liquid-solid interfaces in the Debye layer for hydrophilic and hydrophobic surfaces. We measure velocity profiles and nanotracer concentration and diffusion profiles between 20 and 300 nm from the walls in pressure-driven and electro-osmotic flows. We extract electrostatic and zeta potentials and determine hydrodynamic slip lengths with 10 nm accuracy. The spectacular amplification of the zeta potential resulting from hydrodynamic slippage allows us to clarify for the first time the dynamic origin of the zeta potential. (10.1103/PhysRevLett.101.114503)
DOI : 10.1103/PhysRevLett.101.114503
Tissue deformation modulates Twist expression to determine anterior midgut differentiation in Drosophila embryos
- Desprat Nicolas
- Supatto Willy
- Pouille Philippe-Alexandre
- Beaurepaire Emmanuel
- Farge Emmanuel
Developmental Cell, Elsevier , 2008, 15 (3), pp.470-477 . Mechanical deformations associated with embryonic morphogenetic movements have been suggested to actively participate in the signaling cascades regulating developmental gene expression. Here we develop an appropriate experimental approach to ascertain the existence and the physiological relevance of this phenomenon. By combining the use of magnetic tweezers with in vivo laser ablation, we locally control physiologically relevant deformations in wild-type Drosophila embryonic tissues. We demonstrate that the deformations caused by germ band extension upregulate Twist expression in the stomodeal primordium. We find that stomodeal compression triggers Src42A-dependent nuclear translocation of Armadillo/β-catenin, which is required for Twist mechanical induction in the stomodeum. Finally, stomodeal-specific RNAi-mediated silencing of Twist during compression impairs the differentiation of midgut cells, resulting in larval lethality. These experiments show that mechanically induced Twist upregulation in stomodeal cells is necessary for subsequent midgut differentiation. DEVBIO (10.1016/j.devcel.2008.07.009)
DOI : 10.1016/j.devcel.2008.07.009