Articles

Xanthines Studied via Femtosecond Fluorescence Spectroscopy
- Changenet-Barret P.
- Kovács L.
- Markovitsi D.
- Gustavsson T.
Molecules, MDPI , 2016, 21 (12), pp.1668 . Xanthines represent a wide class of compounds closely related to the DNA bases adenine and guanine. Ubiquitous in the human body, they are capable of replacing natural bases in double helices and give rise to four-stranded structures. Although the use of their fluorescence for analytical purposes was proposed, their fluorescence properties have not been properly characterized so far. The present paper reports the first fluorescence study of xanthine solutions relying on femtosecond spectroscopy. Initially, we focus on 3-methylxanthine, showing that this compound exhibits non-exponential fluorescence decays with no significant dependence on the emission wavelength. The fluorescence quantum yield (3 × 10−4) and average decay time (0.9 ps) are slightly larger than those found for the DNA bases. Subsequently, we compare the dynamical fluorescence properties of seven mono-, di- and tri-methylated derivatives. Both the fluorescence decays and fluorescence anisotropies vary only weakly with the site and the degree of methylation. These findings are in line with theoretical predictions suggesting the involvement of several conical intersections in the relaxation of the lowest singlet excited state. (10.3390/molecules21121668)
DOI : 10.3390/molecules21121668
The Dynamics Behind the Affinity: Controlling Heme-Gas Affinity via Geminate Recombination and Heme Propionate Conformation in the NO Carrier Cytochrome c ′
- Andrew Colin R.
- Petrova Olga N.
- Lamarre Isabelle
- Lambry Jean-Christophe
- Rappaport Fabrice
- Négrerie Michel
ACS Chemical Biology, American Chemical Society , 2016, 11 (11), pp.3191 - 3201 . Nitric oxide (NO) sensors are heme proteins which may also bind CO and O2. Control of heme-gas affinity and their discrimination are achieved by the structural properties and reactivity of the heme and its distal and proximal environments, leading to several energy barriers. In the bacterial NO sensor cytochrome c′ from Alcaligenes xylosoxidans (AXCP), the single Leu16Ala distal mutation boosts the affinity for gas ligands by a remarkable 106–108-fold, transforming AXCP from one of the lowest affinity gas binding proteins to one of the highest. Here, we report the dynamics of diatomics after photodissociation from wild type and L16A-AXCP over 12 orders of magnitude in time. For the L16A variant, the picosecond geminate rebinding of both CO and NO appears with an unprecedented 100% yield, and no exit of these ligands from protein to solvent could be observed. Molecular dynamic simulations saliently demonstrate that dissociated CO stays within 4 Å from Fe2+, in contrast to wild-type AXCP. The L16A mutation confers a heme propionate conformation and docking site which traps the diatomics, maximizing the probability of recombination and directly explaining the ultrahigh affinities for CO, NO, and O2. Overall, our results point to a novel mechanism for modulating heme-gas affinities in proteins. (10.1021/acschembio.6b00599)
DOI : 10.1021/acschembio.6b00599
Unusual Dynamics of Ligand Binding to the Heme Domain of the Bacterial CO Sensor Protein RcoM-2
- Bouzhir-Sima Latifa
- Motterlini Roberto
- Gross Julia
- Vos Marten H.
- Liebl Ursula
Journal of Physical Chemistry B, American Chemical Society , 2016, 120 (41), pp.10686 - 10694 . The aerobic Gram-negative bacterium Burkholderia xenovorans expresses two highly homologous carbon monoxide (CO)-responsive transcriptional regulators, RcoM-1 and RcoM-2, which display extraordinarily high CO affinities, even under oxygenic conditions. To gain insight into the origin and perspectives of this feature, we characterized the ligand-binding properties of the N-terminal, heme-binding Per/Arnt/Sim sensor domain of RcoM-2 by time-resolved spectroscopy. We show that upon photodissociation of the heme–ligand bond, CO geminately rebinds to the heme with picosecond time constants and more than 99% rebinding yield, an unprecedented property of native heme proteins. Remarkably, the rebinding kinetics speeds up when the protein motions are slowed by cooling or solvent viscosity. This indicates that the origin of the observed efficient rebinding is a protein-imposed CO configuration in the heme pocket that is highly favorable for binding, a feature strongly in contrast to that of hemoglobins. The binding of CO to the ferrous heme from the solvent requires dissociation of the methionine axial heme ligand. From the kinetics of ligand binding and the extreme stability of the CO complex, we deduce that the dissociation constant for CO is lower than 100 pM. Finally, we show that when the ferric complex is exposed to CO gas or a CO-releasing molecule under oxygenic conditions formation of the ferrous carbonyl complex can occur on a time scale of minutes in the presence of a redox mediator. These findings pave the way for possible applications of the RcoM-2 heme domain as a CO sensor and/or scavenger. (10.1021/acs.jpcb.6b08160)
DOI : 10.1021/acs.jpcb.6b08160
Thermal stability and binding energetics of thymidylate synthase ThyX
- Krumova Sashka
- Todinova Svetla
- Tileva Milena
- Bouzhir-Sima Latifa
- Vos Marten H.
- Liebl Ursula
- Taneva Stefka G.
International Journal of Biological Macromolecules, Elsevier , 2016, 91, pp.560 - 567 . The bacterial thymidylate synthase ThyX is a multisubstrate flavoenzyme that takes part in the de novo synthesis of thymidylate in a variety of microorganisms. Herein we study the effect of FAD and dUMP binding on the thermal stability of wild type (WT) ThyX from the mesophilic Paramecium bursaria chlorella virus-1 (PBCV-1) and from the thermophilic bacterium Thermotoga maritima (TmThyX), and from two variants of TmThyX, Y91F and S88W, using differential scanning calorimetry. The energetics underlying these processes was characterized by isothermal titration calorimetry. The PBCV-1 protein is significantly less stable against the thermal challenge than the TmThyX WT. FAD exerted stabilizing effect greater for PBCV-1 than for TmThyX and for both mutants, whereas binding of dUMP to FAD-loaded proteins stabilized further only TmThyX. Different thermodynamic signatures describe the FAD binding to the WT ThyX proteins. While TmThyX binds FAD with a low μM binding affinity in a process characterized by a favorable entropy change, the assembly of PBCV-1 with FAD is governed by a large enthalpy change opposed by an unfavorable entropy change resulting in a relatively strong nM binding. An enthalpy-driven formation of a high affinity ternary ThyX/FAD/dUMP complex was observed only for TmThyX. (10.1016/j.ijbiomac.2016.05.083)
DOI : 10.1016/j.ijbiomac.2016.05.083
Synthesis, characterization and spectral temperature-dependence of thioglycerol-CdSe nanocrystals
- Ben Brahim Nassim
- Poggi Mélanie
- Haj Mohamed Mohamed
- Ben Chaâbane Rafik
- Haouari Mohamed
- Negrerie Michel
- Ben Ouada Hafedh
Journal of Luminescence, Elsevier , 2016, 177, pp.402 - 408 . Water-soluble CdSe quantum dots (QDs) have been synthesized with thioglycerol as a stabilizer through a novel hydrothermal route. The obtained thioglycerol capped CdSe (TG-CdSe) nanocrystals were characterized regarding their morphology and structural, thermal and optical properties. The resulting nanocrystals were synthesized in the cubic structure with a near spherical shape, as confirmed by X-ray diffraction and transmission electron microscopy. Combining transmission electron microscopy imaging and calculations using UV–visible absorption spectrum and X-ray diffraction pattern, the diameter of the synthesized nanocrystals was estimated to 2.26 nm. As confirmed by its Fourier transform IR spectrum, thioglycerol was successfully liganded on the surface of the resulting nanocrystals. Band structure parameters of the TG-CdSe nanoparticles were determined and quantum confinement effect was evidenced by optical absorption, fluorescence and Raman measurements. The thermal properties of the TG-CdSe were explored by thermal gravimetric analysis and differential scanning calorimetry. The temperature dependence of both the absorption and fluorescence spectra in the physiological range makes the TG-CdSe nanocrystals sensitive temperature markers, a property that must be taken into account when developing any probing applications, especially for cellular imaging. (10.1016/j.jlumin.2016.05.026)
DOI : 10.1016/j.jlumin.2016.05.026
Transient Two-Dimensional Infrared Spectroscopy in a Vibrational Ladder
- Kemlin Vincent
- Bonvalet Adeline
- Daniault Louis
- Joffre Manuel
Journal of Physical Chemistry Letters, American Chemical Society , 2016, 7 (17), pp.3377 - 3382 . We report on transient 2D Fourier transform infrared spectroscopy (2DIR) after vibrational ladder climbing induced in the CO-moiety longitudinal stretch of carboxyhemoglobin. The population distribution, spreading up to seven vibrational levels, results in a nonequilibrium 2DIR spectrum evidencing a large number of peaks that can be easily attributed to individual transitions thanks to the anharmonicity of the vibrational potential. We discuss the physical origin of the observed peaks as well as the qualitative behavior of the subsequent dynamics governed by population relaxation in the vibrational ladder. (10.1021/acs.jpclett.6b01535)
DOI : 10.1021/acs.jpclett.6b01535
Efficient second-harmonic imaging of collagen in histological slides using Bessel beam excitation
- Vuillemin Nelly
- Mahou Pierre
- Débarre Delphine
- Gacoin Thierry
- Tharaux Pierre-Louis
- Schanne-Klein Marie-Claire
- Supatto Willy
- Beaurepaire Emmanuel
Scientific Reports, Nature Publishing Group , 2016, 6 (29863 (2016)) . econd-harmonic generation (SHG) is the most specific label-free indicator of collagen accumulation in widespread pathologies such as fibrosis, and SHG-based measurements hold important potential for biomedical analyses. However, efficient collagen SHG scoring in histological slides is hampered by the limited depth-of-field of usual nonlinear microscopes relying on focused Gaussian beam excitation. In this work we analyze theoretically and experimentally the use of Bessel beam excitation to address this issue. Focused Bessel beams can provide an axially extended excitation volume for nonlinear microscopy while preserving lateral resolution. We show that shaping the focal volume has consequences on signal level and scattering directionality in the case of coherent signals (such as SHG) which significantly differ from the case of incoherent signals (two-photon excited fluorescence, 2PEF). We demonstrate extended-depth SHG-2PEF imaging of fibrotic mouse kidney histological slides. Finally, we show that Bessel beam excitation combined with spatial filtering of the harmonic light in wave vector space can be used to probe collagen accumulation more efficiently than the usual Gaussian excitation scheme. These results open the way to SHG-based histological diagnoses. (10.1038/srep29863)
DOI : 10.1038/srep29863
Simultaneous microstructural and mechanical characterization of human corneas at increasing pressure
- Benoit Aurélie
- Latour Gaël
- Marie-Claire Schanne-Klein
- Allain Jean-Marc
Journal of the mechanical behavior of biomedical materials, Elsevier , 2016, 60, pp.93-105 . The cornea, through its shape, is the main contributor to the eye׳s focusing power. Pathological alterations of the cornea strongly affect the eye power. To improve treatments, complex biomechanical models have been developed based on the architecture and mechanical properties of the collagen network in the stroma, the main layer of the cornea. However, direct investigations of the structure of the stroma, as well as its link to the mechanical response, remained limited. We propose here an original set up, associating nonlinear optical imaging and mechanical testing. By using polarization resolved Second Harmonic signals, we simultaneously quantified micrometer (orientation of the collagen lamellae) and nanometer (local disorder within lamellae) scale corneal organization. We showed that the organization of the lamellae changes along the stroma thickness. Then, we measured simultaneously the deformation on the epithelial side of the cornea and the reorientation of the collagen lamellae for increasing intraocular pressure levels, from physiological ones to pathological ones. We showed that the observed deformation is not correlated to initial orientation, but to the reorganization of the lamellae in the stroma. Our results, by providing a direct multi-scale observation, will be useful for the development of more accurate biomechanical model (10.1016/j.jmbbm.2015.12.031)
DOI : 10.1016/j.jmbbm.2015.12.031
Ultrafast Electron Transfer in Complexes of Doxorubicin with Human Telomeric G-Quadruplexes and GC Duplexes Probed by Femtosecond Fluorescence Spectroscopy
- Changenet-Barret P.
- Gustavsson T.
- Markovitsi D.
- Manet I.
ChemPhysChem, Wiley-VCH Verlag , 2016, 17 (9), pp.1264–1272 . Doxorubicin (DOX) is a natural anthracycline widely used in chemotherapy; its combined application as a chemotherapeutic and photodynamic agent has been recently proposed. In this context, understanding the photoinduced properties of DOX complexes with nucleic acids is crucial. Herein, the study of photoinduced electron transfer in DOX–DNA complexes by femtosecond fluorescence spectroscopy is reported. The behaviour of complexes with two model DNA structures, a G-quadruplex (G4) formed by the human telomeric sequence (Tel21) and a d(GC) duplex, is compared. The DOX affinity for these two sequences is similar. Although both 1:1 and 2:1 stoichiometries have been reported for DOX–G4 complexes, only 1:1 complexes form with the duplex. The steady-state absorption indicates a strong binding interaction with the duplex due to drug intercalation between the GC base pairs. In contrast, the interaction of DOX with Tel21 is much weaker and arises from drug binding on the G4 external faces at two independent binding sites. As observed for DOX–d(GC) complexes, fluorescence of the drug in the first binding site of Tel21 exhibits decays within a few picoseconds following a biphasic pattern; this is attributed to the existence of two drug conformations. The fluorescence of the drug in the second binding site of Tel21 shows slower decays within 150 ps. These timescales are consistent with electron transfer from the guanines to the excited drug, as favoured by the lower oxidation potential of the stacked guanines of G4 with respect to those in the duplex. (10.1002/cphc.201501091)
DOI : 10.1002/cphc.201501091
Correlative nonlinear optical microscopy and infrared nanoscopy reveals collagen degradation in altered parchments
- Latour Gaël
- Robinet Laurianne
- Dazzi Alexandre
- Portier François
- Deniset-Besseau Ariane
- Schanne-Klein Marie-Claire
Scientific Reports, Nature Publishing Group , 2016, 6, pp.26344 . This paper presents the correlative imaging of collagen denaturation by nonlinear optical microscopy (NLO) and nanoscale infrared (IR) spectroscopy to obtain morphological and chemical information at different length scales. Such multiscale correlated measurements are applied to the investigation of ancient parchments, which are mainly composed of dermal fibrillar collagen. The main issue is to characterize gelatinization, the ultimate and irreversible alteration corresponding to collagen denaturation to gelatin, which may also occur in biological tissues. Key information about collagen and gelatin signatures is obtained in parchments and assessed by characterizing the denaturation of pure collagen reference samples. A new absorbing band is observed near the amide I band in the IR spectra, correlated to the onset of fluorescence signals in NLO images. Meanwhile, a strong decrease is observed in Second Harmonic signals, which are a structural probe of the fibrillar organization of the collagen at the micrometer scale. NLO microscopy therefore appears as a powerful tool to reveal collagen degradation in a non-invasive way. It should provide a relevant method to assess or monitor the condition of collagen-based materials in museum and archival collections and opens avenues for a broad range of applications regarding this widespread biological material. (10.1038/srep26344)
DOI : 10.1038/srep26344
A Primer on the Bayesian Approach to High-Density Single-Molecule Trajectories Analysis
- El beheiry Mohamed
- Türkcan Silvan
- Richly Maximilian u.
- Triller Antoine
- Alexandrou Antigone
- Dahan Maxime
- Masson Jean-Baptiste
Biophysical Journal, Biophysical Society , 2016, 110 (6), pp.1209 - 1215 . (10.1016/j.bpj.2016.01.018)
DOI : 10.1016/j.bpj.2016.01.018
Ultrafast Spectroscopy Evidence for Picosecond Ligand Exchange at the Binding Site of a Heme Protein: Heme-Based Sensor YddV
- Lambry Jean-Christophe
- Stranava Martin
- Lobato Laura
- Martinkova Marketa
- Shimizu Toru
- Liebl Ursula
- Vos Marten H.
Journal of Physical Chemistry Letters, American Chemical Society , 2016, 7 (1), pp.69-74 . An important question for the functioning of heme proteins is whether different ligands present within the protein moiety can readily exchange with heme-bound ligands. Studying the dynamics of the heme domain of the Escherichia coli sensor protein YddV upon dissociation of NO from the ferric heme by ultrafast spectroscopy, we demonstrate that when the hydrophobic leucine residue in the distal heme pocket is mutated to glycine, in a substantial fraction of the protein water replaces NO as an internal ligand in as fast as ∼4 ps. This process, which is near-barrierless and occurs orders of magnitude faster than the corresponding process in myoglobin, corresponds to a ligand swap of NO with a water molecule present in the heme pocket, as corroborated by molecular dynamics simulations. Our findings provide important new insight into ligand exchange in heme proteins that functionally interact with different external ligands. (10.1021/acs.jpclett.5b02517)
DOI : 10.1021/acs.jpclett.5b02517
Probing the 3D structure of cornea-like collagen liquid crystals with polarization-resolved SHG microscopy
- Teulon Claire
- Tidu Aurélien
- Portier François
- Mosser Gervaise
- Schanne-Klein Marie-Claire
Optics Express, Optical Society of America - OSA Publishing , 2016, 24, pp.16084 - 10 . This work aims at characterizing the three-dimensional organization of liquid crystals composed of collagen, in order to determine the physico-chemical conditions leading to highly organized structures found in biological tissues such as cornea. To that end, we use second-harmonic generation (SHG) microscopy, since aligned collagen structures have been shown to exhibit intrinsic SHG signals. We combine polarization-resolved SHG experiments (P-SHG) with the theoretical derivation of the SHG signal of collagen molecules tilted with respect to the focal plane. Our P-SHG images exhibit striated patterns with variable contrast, as expected from our analytical and numerical calculations for plywood-like nematic structures similar to the ones found in the cornea. This study demonstrates the benefits of P-SHG microscopy for in situ characterization of highly organized biopolymers at micrometer scale, and the unique sensitivity of this nonlinear optical technique to the orientation of collagen molecules. (10.1364/OE.24.016084.v001)
DOI : 10.1364/OE.24.016084.v001
Direct observation of subpicosecond vibrational dynamics in photoexcited myoglobin
- Ferrante C.
- Pontecorvo E.
- Cerullo Giulio
- Vos Marten H.
- Scopigno T.
Nature Chemistry, Nature Publishing Group , 2016 (8), pp.1137–1143 . Determining the initial pathway for ultrafast energy redistribution within biomolecules is a challenge, and haem proteins, for which energy can be deposited locally in the haem moiety using short light pulses, are suitable model systems to address this issue. However, data acquired using existing experimental techniques that fail to combine sufficient structural sensitivity with adequate time resolution have resulted in alternative hypotheses concerning the interplay between energy flow among highly excited vibrational levels and potential concomitant electronic processes. By developing a femtosecond-stimulated Raman set-up, endowed with the necessary tunability to take advantage of different resonance conditions, here we visualize the temporal evolution of energy redistribution over different vibrational modes in myoglobin. We establish that the vibrational energy initially stored in the highly excited Franck–Condon manifold is transferred with different timescales into low- and high-frequency modes, prior to slow dissipation through the protein. These findings demonstrate that a newly proposed mechanism involving the population dynamics of specific vibrational modes settles the controversy on the existence of transient electronic intermediates. (10.1038/nchem.2569)
DOI : 10.1038/nchem.2569
Predictive modeling targets thymidylate synthase ThyX in Mycobacterium tuberculosis
- Djaout Kamel
- Singh Vinayak
- Boum Yap
- Katawera Victoria
- Becker Hubert F.
- Bush Natassja G.
- Hearnshaw Stephen J.
- Pritchard Jennifer E.
- Bourbon Pauline
- Madrid Peter B.
- Maxwell Anthony
- Mizrahi Valerie
- Myllykallio Hannu
- Ekins Sean
Scientific Reports, Nature Publishing Group , 2016, 6, pp.27792 . There is an urgent need to identify new treatments for tuberculosis (TB), a major infectious disease caused by Mycobacterium tuberculosis (Mtb), which results in 1.5 million deaths each year. We have targeted two essential enzymes in this organism that are promising for antibacterial therapy and reported to be inhibited by naphthoquinones. ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the human enzyme. DNA gyrase is a DNA topoisomerase present in bacteria and plants but not animals. The current study set out to understand the structure-activity relationships of these targets in Mtb using a combination of cheminformatics and in vitro screening. Here, we report the identification of new Mtb ThyX inhibitors, 2-chloro-3-(4-methanesulfonylpiperazin-1-yl)-1,4-dihydronaphthalene-1,4-dione) and idebenone, which show modest whole-cell activity and appear to act, at least in part, by targeting ThyX in Mtb. (10.1038/srep27792)
DOI : 10.1038/srep27792
Thioglycerol-functionalized CdSe quantum dots detecting cadmium ions
- Brahim Nassim Ben
- Mohamed Naim Bel Haj
- Echabaane Mosaab
- Haouari Mohamed
- Chaâbane Rafik Ben
- Negrerie Michel
- Ouada Hafedh Ben
Sensors and Actuators B: Chemical, Elsevier , 2015, 220, pp.1346-1353 . Water-soluble CdSe quantum dots (QDs) were synthesized using thioglycerol (TG) as the surface capping agent through a one-step process at low temperature T (100 degrees C). The CdSe quantum dots were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy, UV-visible absorption and fluorescence spectroscopies. These measurements revealed that the TG-capped CdSe QDs possess a high crystalline quality with an average diameter in the range 2.5-2.8 nm and exhibit particular optical properties. The UV-visible absorption of CdSe QDs is enhanced by the addition of cadmium ions, with a simultaneous shift of the edge band (400 nm), while seventeen other tested metal cations have no effect on the absorption of QDs. Moreover, the binding of Cd2+ ions induces a quenching of the fluorescence emission of TG-CdSe QDs. At particular absorption wavelengths, the response is linearly proportional to the cadmium ions concentration ranging from 1.0 to 22 mu M with a detection limit of 0.32 mu M (37 mu g L-1). Based on these optical properties, the TG-CdSe QDs could be used as a highly selective probe for the detection of Cd2+ ions in aqueous solutions, a species highly toxic for cells. (C) 2015 Elsevier B.V. All rights reserved. (10.1016/j.snb.2015.07.049)
DOI : 10.1016/j.snb.2015.07.049
Conservation and Role of Electrostatics in Thymidylate Synthase
- Garg Divita
- Skouloubris Stephane
- Briffotaux Julien
- Myllykallio Hannu
- Wade Rebecca C
Scientific Reports, Nature Publishing Group , 2015 . Conservation of function across families of orthologous enzymes is generally accompanied by conservation of their active site electrostatic potentials. To study the electrostatic conservation in the highly conserved essential enzyme, thymidylate synthase (TS), we conducted a systematic species-based comparison of the electrostatic potential in the vicinity of its active site. Whereas the electrostatics of the active site of TS are generally well conserved, the TSs from minimal organisms do not conform to the overall trend. Since the genomes of minimal organisms have a high thymidine content compared to other organisms, the observation of non-conserved electrostatics was surprising. Analysis of the symbiotic relationship between minimal organisms and their hosts, and the genetic completeness of the thymidine synthesis pathway suggested that TS from the minimal organism Wigglesworthia glossinidia (W.g.b.) must be active. Four residues in the vicinity of the active site of Escherichia coli TS were mutated individually and simultaneously to mimic the electrostatics of W.g.b TS. The measured activities of the E. coli TS mutants imply that conservation of electrostatics in the region of the active site is important for the activity of TS, and suggest that the W.g.b. TS has the minimal activity necessary to support replication of its reduced genome. The electrostatic potential of a protein plays a crucial role in steering ligands to their binding sites, and orienting them correctly for binding 1. In enzymes, the active site electrostatic potential is important for stabilizing the transition state and thereby catalyzing the reaction 2. Therefore, conservation of protein function across a protein family is often accompanied by conservation of the electrostatic potential in the active site region, even though the rest of the protein may lack a conserved electrostatic potential 3,4. Consequently, comparison of protein electrostatic potentials has been employed as a tool to predict protein function and to derive similarities in protein function across protein families 5–7. Optimizing the electrostatic complementarity between a ligand and the binding site of a protein is also an important aspect in drug design 8,9 and may provide a route to gain target selectivity 10 . (10.1038/srep17356)
DOI : 10.1038/srep17356
DNA replication restart and cellular dynamics of Hef helicase/nuclease protein in Haloferax volcanii
- Lestini Roxane
- Delpech Floriane
- Myllykallio Hannu
Biochimie, Elsevier , 2015, 118, pp.254-263 . Understanding how frequently spontaneous replication arrests occur and how archaea deal with these arrests are very interesting and challenging research topics. Here we will described how genetic and imaging studies have revealed the central role of the archaeal helicase/nuclease Hef belonging to the XPF/MUS81/FANCM family of endonucleases in repair of arrested replication forks. Special focus will be on description of a recently developed combination of genetic and imaging tools to study the dynamic localization of a functional Hef::GFP (Green. Fluorescent Protein) fusion protein in the living cells of halophilic archaea Haloferax volcanii. As Archaea provide an excellent and unique model for understanding how DNA replication is regulated to allow replication of a circular DNA molecule either from single or multiple replication origins, we will also summarize recent studies that have revealed peculiar features regarding DNA replication, particularly in halophilic archaea. We strongly believe that fundamental knowledge of our on-going studies will shed light on the evolutionary history of the DNA replication machinery and will help to establish general rules concerning replication restart and the key role of recombination proteins not only in bacteria, yeast and higher eukaryotes but also in archaea. (C) 2015 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). (10.1016/j.biochi.2015.07.022)
DOI : 10.1016/j.biochi.2015.07.022
Arbitrary-detuning asynchronous optical sampling with amplified laser systems
- Antonucci Laura
- Bonvalet Adeline
- Solinas Xavier
- Daniault Louis
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2015, 23 (21), pp.27931-27940 . We demonstrate that Arbitrary-Detuning ASynchronous OPtical Sampling (AD-ASOPS) makes possible multiscale pump-probe spectroscopy with time delays spanning from picosecond to millisecond. The implementation on pre-existing femtosecond amplifiers seeded by independent free-running oscillators is shown to be straightforward. The accuracy of the method is determined by comparison with spectral interferometry, providing a distribution with a standard deviation ranging from 0.31 to 1.7 ps depending on experimental conditions and on the method used to compute the AD-ASOPS delays. (C) 2015 Optical Society of America (10.1364/OE.23.027931)
DOI : 10.1364/OE.23.027931
Large-scale live imaging of adult neural stem cells in their endogenous niche
- Dray Nicolas
- Bedu Sébastien
- Vuillemin Nelly
- Alunni Alessandro
- Coolen Marion
- Krecsmarik Monika
- Supatto Willy
- Beaurepaire Emmanuel
- Bally-Cuif Laure
Development (Cambridge, England), Company of Biologists , 2015, 142 (20), pp.3592-3600 . Live imaging of adult neural stem cells (aNSCs) in vivo is a technical challenge in the vertebrate brain. Here, we achieve long-term imaging of the adult zebrafish telencephalic neurogenic niche and track a population of >1000 aNSCs over weeks, by taking advantage of fish transparency at near-infrared wavelengths and of intrinsic multiphoton landmarks. This methodology enables us to describe the frequency, distribution and modes of aNSCs divisions across the entire germinal zone of the adult pallium, and to highlight regional differences in these parameters. (10.1242/dev.123018)
DOI : 10.1242/dev.123018
High-power, widely tunable, room-temperature picosecond optical parametric oscillator based on cylindrical 5%MgO:PPLN
- Chaitanya Kumar S.
- Wei Junxiong
- Debray J.
- Kemlin Vincent
- Boulanger B.
- Ishizuki Hideki
- Taira T.
- Ebrahim-Zadeh M.
Optics Letters, Optical Society of America - OSA Publishing , 2015, 40 (16), pp.3897-3900 . We report a high-power picosecond optical parametric oscillator (OPO) based on cylindrical MgO:PPLN synchronously pumped by an Yb-fiber laser. The singly resonant OPO is tunable in the near-infrared signal across 1413–1900 nm and mid-infrared idler over 2418–4307 nm by angle tuning of the crystal at room temperature. With non-optimized output coupling of ∼10%, the OPO simultaneously delivers 2.4 W of signal at 1664 nm and 1.7 W of idler at 2950 nm at an overall extraction efficiency of ∼45% with high beam-pointing stability <30 μrad and <14 μrad for the signal and idler, respectively. The generated signal and idler exhibit passive power stability better than 1% rms and 0.8% rms over 15 h, respectively, in high beam quality with TEM00 profile. The extracted signal pulses from the OPO have duration of 15.2 ps with a spectral bandwidth of 0.7 nm, corresponding to a time-bandwidth product of ΔvΔtau∼1.2. (10.1364/OL.40.003897)
DOI : 10.1364/OL.40.003897
Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity
- Fojtikova Veronika
- Stranava Martin
- Vos Marten H.
- Liebl Ursula
- Hranicek Jakub
- Kitanishi Kenichi
- Shimizu Toru
- Martinkova Marketa
Biochemistry, American Chemical Society , 2015, 54 (32), pp.5017–5029 . The globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction’s kinetics. The kcat values of the heme Fe(III)-OH–, Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1–1.2 min–1, and their KmATP values were 18.9–35.4 μM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min–1 but a very high KmATP value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and KmATP values of 0.4 min–1 and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and KmATP values of 0.3 min–1 and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme’s catalytic activity because they modulate its ATP binding affinity and thus change its kcat/KmATP value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed (10.1021/acs.biochem.5b00517)
DOI : 10.1021/acs.biochem.5b00517
Optimization of Picrosirius red staining protocol to determine collagen fiber orientations in vaginal and uterine cervical tissues by Mueller polarized microscopy
- Nazac André
- Bancelin Stéphane
- Teig Benjamin
- Haj Ibrahim Bicher
- Fernandez Hervé
- Schanne-Klein Marie-Claire
- de Martino Antonello
Microscopy Research and Technique, Wiley , 2015, 78 (8), pp.723-730 . Polarized microscopy provides unique information on anisotropic samples. In its most complete implementation, namely Mueller microscopy, this technique is well suited for the visualization of fibrillar proteins orientations, with collagen in the first place. However, the intrinsic optical anisotropy of unstained tissues has to be enhanced by Picrosirius Red (PR) staining to enable Mueller measurements. In this work, we compared the orientation mapping provided by Mueller and second harmonic generation (SHG) microscopies on PR stained samples of vaginal and uterine cervix tissues. SHG is a multiphoton technique that is highly specific to fibrillar collagen, and was taken as the gold standard for its visualization. We showed that Mueller microscopy can be safely used to determine collagen orientation in PR stained cervical tissue. In contrast, in vaginal samples, Mueller microscopy revealed orientations not only of collagen but also of other anisotropic structures. Thus PR is not fully specific to collagen, which necessitates comparison to SHG microscopy in every type of tissue. In addition to this study of PR specificity, we determined the optimal values of the staining parameters. We found that staining times of 5min, and sample thicknesses of 5 mu m were sufficient in cervical and vaginal tissues. Microsc. Res. Tech. 78:723-730, 2015. (c) 2015 Wiley Periodicals, Inc. (10.1002/jemt.22530)
DOI : 10.1002/jemt.22530
Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease
- Camus S. M.
- de Moraes J. A.
- Bonnin P.
- Abbyad P.
- Le Jeune S.
- Lionnet F.
- Loufrani L.
- Grimaud L.
- Lambry J.-C.
- Charue D.
- Kiger L.
- Renard J.-M.
- Larroque C.
- Le Clesiau H.
- Tedgui A.
- Bruneval P.
- Barja-Fidalgo C.
- Alexandrou A.
- Tharaux P.-L.
- Boulanger C. M.
- Blanc-Brude O. P.
Blood, American Society of Hematology , 2015, 125 (24), pp.3805-3814 . Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that hemeladen MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of hemeladen MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD. (10.1182/blood-2014-07-589283)
DOI : 10.1182/blood-2014-07-589283
Ultrafast Dynamics of Carboxy-Hemoglobin: Two-Dimensional Infrared Spectroscopy Experiments and Simulations
- Falvo Cyril
- Daniault Louis
- Vieille Thibault
- Kemlin Vincent
- Lambry Jean-Christophe
- Meier Christoph
- Vos Marten H.
- Bonvalet Adeline
- Joffre Manuel
Journal of Physical Chemistry Letters, American Chemical Society , 2015, 6 (12), pp.2216–2222 . This Letter presents a comparison between experimental and simulated 2D mid-infrared spectra of carboxy-hemoglobin in the spectral region of the carbon monoxide stretching mode. The simulations rely on a fluctuating potential energy surface that includes both the effect of heme and the protein surroundings computed from molecular dynamics simulations. A very good agreement between theory and experiment is obtained with no adjustable parameters. The simulations show that the effect of the distal histidine through the hydrogen bond is strong and is directly responsible for the slow decay of the frequency–frequency correlation function on a 10 ps time scale. This study confirms that fluctuations in carboxy-hemoglobin are more inhomogeneous than those in the more frequently studied carboxy-myoglobin. The comparison between simulations and experiments brings valuable information on the complex relation between protein structure and spectral diffusion. (10.1021/acs.jpclett.5b00811)
DOI : 10.1021/acs.jpclett.5b00811