Articles

Early processes in heme-based CO-sensing proteins
- Vos Marten H
- Salman Mayla
- Liebl Ursula
Frontiers in Molecular Biosciences, Frontiers Media , 2022, 9, pp.1046412 . Carbon monoxide has been recognized relatively recently as signaling molecule, and only very few dedicated natural CO sensor proteins have been identified so far. These include in particular heme-based transcription factors: the bacterial sensor proteins CooA and RcoM. In these 6-coordinated systems, exchange between an internal protein residue and CO as a heme ligand in the sensor domain affects the properties of the DNA-binding domain. Using light to dissociate heme-ligand bonds can in principle initiate this switching process. We review the efforts to use this method to investigate early processes in ligand switching and signaling, with an emphasis on the CO-"trapping" properties of the heme cavity. These features are unusual for most heme proteins, but common for heme-based CO sensors. (10.3389/fmolb.2022.1046412)
DOI : 10.3389/fmolb.2022.1046412
Inline amplification of mid-infrared intrapulse difference frequency generation
- Bournet Q.
- Jonusas M.
- Zheng A.
- Guichard F.
- Natile M.
- Zaouter Y.
- Joffre M.
- Bonvalet A.
- Druon F.
- Hanna Marc
- Georges P.
Optics Letters, Optical Society of America - OSA Publishing , 2022, 47 (19), pp.4885 . We demonstrate an ultrafast mid-infrared source architecture that implements both intrapulse difference frequency generation (iDFG) and further optical parametric amplification (OPA), in an all-inline configuration. The source is driven by a nonlinearly compressed high-energy Yb-doped-fiber amplifier delivering 7.4 fs pulses at a central wavelength of 1030 nm, at a repetition rate of 250 kHz. It delivers 1 µJ, 73 fs pulses at a central wavelength of 8 µm, tunable over more than one octave. By enrolling all the pump photons in the iDFG process and recycling the long wavelength pump photons amplified in the iDFG in the subsequent OPA, we obtain an unprecedented overall optical efficiency of 2%. These performances, combining high energy and repetition rate in a very simple all-inline setup, make this technique ideally suited for a growing number of applications, such as high harmonic generation in solids or two-dimensional infrared spectroscopy experiments. (10.1364/OL.467792)
DOI : 10.1364/OL.467792
The TPLATE complex mediates membrane bending during plant clathrin–mediated endocytosis
- Johnson Alexander
- Dahhan Dana A
- Gnyliukh Nataliia
- Kaufmann Walter A
- Zheden Vanessa
- Costanzo Tommaso
- Mahou Pierre
- Hrtyan Mónika
- Wang Jie
- Aguilera-Servin Juan
- van Damme Daniël
- Beaurepaire Emmanuel
- Loose Martin
- Bednarek Sebastian Y
- Friml Jiří
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2021, 118 (51) . Clathrin-mediated endocytosis is the major route of entry of cargos into cells and thus underpins many physiological processes. During endocytosis, an area of flat membrane is remodeled by proteins to create a spherical vesicle against intracellular forces. The protein machinery which mediates this membrane bending in plants is unknown. However, it is known that plant endocytosis is actin independent, thus indicating that plants utilize a unique mechanism to mediate membrane bending against high-turgor pressure compared to other model systems. Here, we investigate the TPLATE complex, a plant-specific endocytosis protein complex. It has been thought to function as a classical adaptor functioning underneath the clathrin coat. However, by using biochemical and advanced live microscopy approaches, we found that TPLATE is peripherally associated with clathrin-coated vesicles and localizes at the rim of endocytosis events. As this localization is more fitting to the protein machinery involved in membrane bending during endocytosis, we examined cells in which the TPLATE complex was disrupted and found that the clathrin structures present as flat patches. This suggests a requirement of the TPLATE complex for membrane bending during plant clathrin-mediated endocytosis. Next, we used in vitro biophysical assays to confirm that the TPLATE complex possesses protein domains with intrinsic membrane remodeling activity. These results redefine the role of the TPLATE complex and implicate it as a key component of the evolutionarily distinct plant endocytosis mechanism, which mediates endocytic membrane bending against the high-turgor pressure in plant cells. Copyright © 2021 the Author(s). Published by PNAS. (10.1073/pnas.2113046118)
DOI : 10.1073/pnas.2113046118
hnRNPA1/UP1 Unfolds KRAS G-Quadruplexes and Feeds a Regulatory Axis Controlling Gene Expression
- Ferino Annalisa
- Marquevielle Julien
- Choudhary Himanshi
- Cinque Giorgio
- Robert Coralie
- Bourdoncle Anne
- Picco Raffaella
- Mergny Jean-Louis
- Salgado Gilmar
- Xodo Luigi
ACS Omega, ACS Publications , 2021, 6 (49), pp.34092-34106 . (10.1021/acsomega.1c05538)
DOI : 10.1021/acsomega.1c05538
Exploring Sequence Space to Design Controllable G-Quadruplex Topology Switches
- Chen Jielin
- Cheng Mingpan
- Stadlbauer Petr
- Šponer Jiří
- Mergny Jean-Louis
- Ju Huangxian
- Zhou Jun
CCS Chemistry, Chinese Chemical Society , 2021, pp.3232 - 3246 . As nonclassical nucleic acid structures, G-quadruplexes (G4s) not only play important roles in gene regulation and stability maintenance, but are also widely used in nanotechnology. Structural diversity is one of the main factors explaining the popularity of G4s, but a comprehensive and integrated study of different factors determining G4 structural versatility is currently lacking. Herein, starting from a common G4 sequence, (G 3 T) 3 G 3 , as the parent chain, and then taking advantage of G4 versatility, we present a variety of strategies to control G4 structure, based on the regulation of loop length and flanking sequences, cation (type and concentration), and molecular crowding. These strategies allow us to convert the G4 topology from parallel to hybrid, to antiparallel, and then back to parallel. Such structural diversity reveals the coding regulation ability of G4 structures, with potential applications in nanotechnology. (10.31635/ccschem.021.202101357)
DOI : 10.31635/ccschem.021.202101357
The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology
- Chen Jielin
- Cheng Mingpan
- Salgado Gilmar F
- Stadlbauer Petr
- Zhang Xiaobo
- Amrane Samir
- Guédin Aurore
- He Fangni
- Šponer Jiří
- Ju Huangxian
- Mergny Jean-Louis
- Zhou Jun
Nucleic Acids Research, Oxford University Press , 2021, 49 (16), pp.9548-9559 . Abstract Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the ‘flanking effect’ in this work. This ‘flanking effect’ was more pronounced when nucleotides were added at the 5′-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5′-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context. (10.1093/nar/gkab681)
DOI : 10.1093/nar/gkab681
Phase-modulated rapid-scanning fluorescence-detected two-dimensional electronic spectroscopy
- Agathangelou Damianos
- Javed Ariba
- Sessa Francesco
- Solinas Xavier
- Joffre Manuel
- Ogilvie Jennifer
The Journal of Chemical Physics, American Institute of Physics , 2021, 155 (9), pp.094201 . (10.1063/5.0057649)
DOI : 10.1063/5.0057649
Multiscale conformational dynamics probed by time‐resolved circular dichroism from seconds to picoseconds
- Hache François
- Changenet Pascale
Chirality, Wiley , 2021, 33 (11), pp.747-757 . Time-resolved circular dichroism has been developed for a few decades to investigate rapid conformational changes in (bio)molecules. In our group, we have come up with several experimental set-ups allowing us to study pico-nanosecond local phenomena in molecular systems as well as much slower effects occurring in proteins and DNA in the folding processes. After an overview of the worldwide realizations in this domain, we present emblematic experiments that we have carried out, spanning time domain from picoseconds to seconds. (10.1002/chir.23359)
DOI : 10.1002/chir.23359
Novel G-quadruplex prone sequences emerge in the complete assembly of the human X chromosome
- Bohálová Natália
- Mergny Jean-Louis
- Brázda Václav
Biochimie, Elsevier , 2021, 191, pp.87-90 . (10.1016/j.biochi.2021.09.004)
DOI : 10.1016/j.biochi.2021.09.004
Luminescent lanthanide nanoparticle-based imaging enables ultra-sensitive, quantitative and multiplexed <i>in vitro</i> lateral flow immunoassays
- Mousseau F.
- Féraudet-Tarisse C.
- Simon S.
- Gacoin T.
- Alexandrou A.
- Bouzigues C I
Nanoscale, Royal Society of Chemistry , 2021, 13 (35), pp.14814 - 14824 . Lateral Flow Assays (LFAs) have been extensively used on-site to rapidly detect analytes, possibly in complex media. However, standard gold nanoparticle-based LFAs lack sensitivity and cannot provide quantitative measurements with high accuracy. To overcome these limitations, we image lanthanidedoped nanoparticles (YVO 4 :Eu 40%) as new luminescent LFA probes, using a homemade reader coupled to a smartphone and propose an original image analysis allowing strip quantification regardless of the shape of the test band signal. This method is demonstrated for the detection of staphylococcal enterotoxins SEA, SEG, SEH, and SEI. A systematic comparison to state-of-the-art gold nanoparticle-based LFA revealed an analytical sensitivity enhancement of at least one order of magnitude. We furthermore provided measurements of absolute toxin concentration over two orders of magnitude and demonstrated simultaneous quantitative detection of multiple toxins with unaltered sensitivity. In particular, we reached concentrations 100 times lower than the ones reported in the literature for on-site multiplexed LFA targeting enterotoxins. Altogether, these results highlight that our luminescent nanoparticle-based method provides a powerful and versatile on-site framework to detect multiple biomolecules with sensitivity approaching that obtained by ELISA. This paves the way to a change of paradigm in the field of analytical immunoassays by providing fast in situ quantitative high sensitivity detection of biomarkers or pathogens. (10.1039/d1nr03358a)
DOI : 10.1039/d1nr03358a
High precision dual-modulation differential terahertz ATR sensor for liquid measurements
- Zheng Xiujun
- Gevart Thomas
- Gallot Guilhem
Optics Letters, Optical Society of America - OSA Publishing , 2021, 46 (16), pp.4045-4048 . We describe a highly sensitive and stable quantum-cascade laser-based attenuated total reflection (ATR) terahertz sensor for the detection of very low concentration solutions, using a dual-modulation differential approach and ATR geometry. This sensor offers a very high dynamic range and a long-term stability of 40 dB, which extends the potential of terahertz radiation for the analysis of liquid and biological samples. The performance is illustrated by measurements on standard solutions of ions, sugars, and proteins, for concentrations down to 1 M.
Nanoaggregate-forming lipid-conjugated AS1411 aptamer as a promising tumor-targeted delivery system of anticancer agents in vitro
- Carvalho Josué
- Lopes-Nunes Jéssica
- Vialet Brune
- Rosado Tiago
- Gallardo Eugenia
- Vale João
- Eloy Catarina
- Ferreira Sofia
- Palmeira-De-Oliveira Rita
- Campello Maria Paula Cabral
- Paulo António
- Barthélémy Philippe
- Mergny Jean-Louis
- Salgado Gilmar
- Queiroz João
- Ellington Andrew
- Cruz Carla
Nanomedicine: Nanotechnology, Biology and Medicine, Elsevier , 2021, 36 (13), pp.102429 . Abstract The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds. (10.1016/j.nano.2021.102429)
DOI : 10.1016/j.nano.2021.102429
Probing living cells permeabilization dynamics by terahertz attenuated total reflection
- Gallot Guilhem
Proceedings of SPIE, the International Society for Optical Engineering, SPIE, The International Society for Optical Engineering , 2021 . Using attenuated total reflection (ATR) in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of live epithelial cells by saponin detergent and after electropermeabilization. The origin of the contrast observed between cells and culture medium is addressed by both experimental and theoretical approaches, and gives access to permeabilization dynamics of live cells in real time. We show that terahertz modalities are more sensitive than fluorescence microscopy which is the reference optical technique for electropermeabilization. We propose analytical models for the influx and efflux of non-permeant molecules through permeabilized cell membranes. (10.1117/12.2596166)
DOI : 10.1117/12.2596166
The role of G-Quadruplex DNA in Paraspeckle formation in cancer
- Bhatt Uditi
- Kretzmann Amy
- Guédin Aurore
- Ou Arnold
- Kobelke Simon
- Bond Charles
- Evans Cameron
- Hurley Laurence
- Mergny Jean-Louis
- Iyer K. Swaminathan
- Fox Archa
- Smith Nicole
Biochimie, Elsevier , 2021 . (10.1016/j.biochi.2021.07.008)
DOI : 10.1016/j.biochi.2021.07.008
Noninvasive quantitative assessment of collagen degradation in parchments by polarization-resolved SHG microscopy
- Schmeltz Margaux
- Robinet Laurianne
- Heu-Thao Sylvie
- Sintès Jean-Marc
- Teulon Claire
- Ducourthial Guillaume
- Mahou Pierre
- Schanne-Klein Marie-Claire
- Latour Gaël
Science Advances, American Association for the Advancement of Science (AAAS) , 2021, 7 (29), pp.eabg1090 . Nondestructive and noninvasive investigation techniques are highly sought-after to establish the degradation state of historical parchments, which is up to now assessed by thermal techniques that are invasive and destructive. We show that advanced nonlinear optical (NLO) microscopy enables quantitative in situ mapping of parchment degradation at the micrometer scale. We introduce two parameters that are sensitive to different degradation stages: the ratio of two-photon excited fluorescence to second harmonic generation (SHG) signals probes severe degradation, while the anisotropy parameter extracted from polarization-resolved SHG measurements is sensitive to early degradation. This approach is first validated by comparing NLO quantitative parameters to thermal measurements on artificially altered contemporary parchments. We then analyze invaluable parchments from the Middle Ages and show that we can map their conservation state and assess the impact of a restoration process. NLO quantitative microscopy should therefore help to identify parchments most at risk and optimize restoration methods. (10.1126/sciadv.abg1090)
DOI : 10.1126/sciadv.abg1090
Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula
- Boutillon Arthur
- Escot Sophie
- David Nicolas B.
Journal of visualized experiments : JoVE, JoVE , 2021 (173) . Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns. (10.3791/62815)
DOI : 10.3791/62815
Folding Dynamics of DNA G-Quadruplexes Probed by Millisecond Temperature Jump Circular Dichroism
- Laouer K
- Schmid M
- Wien F
- Changenet Pascale
- Hache François
Journal of Physical Chemistry B, American Chemical Society , 2021 . (10.1021/acs.jpcb.1c01993)
DOI : 10.1021/acs.jpcb.1c01993
Modeling nonlinear microscopy near index-mismatched interfaces
- Morizet Josephine
- Sartorello Giovanni
- Dray Nicolas
- Stringari Chiara
- Beaurepaire Emmanuel
- Olivier Nicolas
Optica, Optical Society of America - OSA Publishing , 2021, 8 (7), pp.944 . Nonlinear microscopy is widely used to characterize thick, optically heterogeneous biological samples. While quantitative image analysis requires accurately describing the contrast mechanisms at play, the majority of established numerical models neglect the influence of field distortion caused by sample heterogeneity near focus. In this work, we show experimentally and numerically that finite-difference time-domain (FDTD) methods are applicable to model focused fields interactions in the presence of heterogeneities, typical of nonlinear microscopy. We analyze the ubiquitous geometry of a vertical interface between index-mismatched media (water, glass, and lipids) and consider the cases of two-photon-excited fluorescence (2PEF), third-harmonic generation (THG) and polarized THG contrasts. We show that FDTD simulations can accurately reproduce experimental images obtained on model samples and in live adult zebrafish, in contrast with previous models neglecting field distortions caused by index mismatch at the micrometer scale. Accounting for these effects appears to be particularly critical when interpreting coherent and polarization-resolved microscopy data. (10.1364/OPTICA.421257)
DOI : 10.1364/OPTICA.421257
Quantitative structural imaging of keratoconic corneas using polarization-resolved SHG microscopy
- Raoux Clothilde
- Schmeltz Margaux
- Bied Marion
- Alnawaiseh Maged
- Hansen Uwe
- Latour Gaël
- Schanne-Klein Marie-Claire
Biomedical optics express, Optical Society of America - OSA Publishing , 2021, 12 . The human cornea is mainly composed of collagen fibrils aligned together within stacked lamellae. This lamellar structure can be affected in pathologies such as keratoconus, which is characterized by progressive corneal thinning and local steepening. In this study, we use polarization-resolved second harmonic generation (P-SHG) microscopy to characterize 8 control and 6 keratoconic human corneas. Automated processing of P-SHG images of transverse sections provides the collagen orientation in every pixel with sub-micrometer resolution. Series of P-SHG images recorded in the most anterior region of the stroma evidence sutural lamellae inclined at 22°± 5°to the corneal surface, but show no significant difference between control and keratoconic corneas. In contrast, series of P-SHG images acquired along the full thickness of the stroma show a loss of order in the lamellar structure of keratoconic corneas, in agreement with their defective mechanical properties. This structural difference is analyzed quantitatively by computing the entropy and the orientation index of the collagen orientation distribution and significant differences are obtained along the full thickness of the stroma. This study shows that P-SHG is an effective tool for automatic quantitative analysis of structural defects of human corneas and should be applied to other collagen-rich tissues. (10.1364/boe.426145)
DOI : 10.1364/boe.426145
Additive CHARMM36 Force Field for Nonstandard Amino Acids
- Croitoru Anastasia
- Park Sang-Jun
- Kumar Anmol
- Lee Jumin
- Im Wonpil
- Mackerell Alexander
- Aleksandrov Alexey
Journal of Chemical Theory and Computation, American Chemical Society , 2021, 17 (6), pp.3554-3570 . (10.1021/acs.jctc.1c00254)
DOI : 10.1021/acs.jctc.1c00254
Exposure to the Methylselenol Precursor Dimethyldiselenide Induces a Reductive Endoplasmic Reticulum Stress in Saccharomyces cerevisiae
- Dauplais Marc
- Mahou Pierre
- Plateau Pierre
- Lazard Myriam
International Journal of Molecular Sciences, MDPI , 2021, 22 (11), pp.5467 . Methylselenol (MeSeH) is a major cytotoxic metabolite of selenium, causing apoptosis in cancer cells through mechanisms that remain to be fully established. Previously, we demonstrated that, in Saccharomyces cerevisiae, MeSeH toxicity was mediated by its metabolization into selenomethionine by O-acetylhomoserine (OAH)-sulfhydrylase, an enzyme that is absent in higher eukaryotes. In this report, we used a mutant met17 yeast strain, devoid of OAH- sulfhydrylase activity, to identify alternative targets of MeSeH. Exposure to dimethyldiselenide (DMDSe), a direct precursor of MeSeH, caused an endoplasmic reticulum (ER) stress, as evidenced by increased expression of the ER chaperone Kar2p. Mutant strains (∆ire1 and ∆hac1) unable to activate the unfolded protein response were hypersensitive to MeSeH precursors but not to selenomethionine. In contrast, deletion of YAP1 or SKN7, required to activate the oxidative stress response, did not affect cell growth in the presence of DMDSe. ER maturation of newly synthesized carboxypeptidase Y was impaired, indicating that MeSeH/DMDSe caused protein misfolding in the ER. Exposure to DMDSe resulted in induction of the expression of the ER oxidoreductase Ero1p with concomitant reduction of its regulatory disulfide bonds. These results suggest that MeSeH disturbs protein folding in the ER by generating a reductive stress in this compartment. (10.3390/ijms22115467)
DOI : 10.3390/ijms22115467
GeNePy3D: a quantitative geometry python toolbox for bioimaging
- Phan Minh-Son
- Chessel Anatole
F1000Research, Faculty of 1000 , 2021 . The advent of large-scale fluorescence and electronic microscopy techniques along with maturing image analysis is giving life sciences a deluge of geometrical objects in 2D/3D(+t) to deal with. These objects take the form of large scale, localised, precise, single cell, quantitative data such as cells’ positions, shapes, trajectories or lineages, axon traces in whole brains atlases or varied intracellular protein localisations, often in multiple experimental conditions. The data mining of those geometrical objects requires a variety of mathematical and computational tools of diverse accessibility and complexity. Here we present a new Python library for quantitative 3D geometry called GeNePy3D which helps handle and mine information and knowledge from geometric data, providing a unified application programming interface (API) to methods from several domains including computational geometry, scale space methods or spatial statistics. By framing this library as generically as possible, and by linking it to as many state-of-the-art reference algorithms and projects as needed, we help render those often specialist methods accessible to a larger community. We exemplify the usefulness of the GeNePy3D toolbox by re-analysing a recently published whole-brain zebrafish neuronal atlas, with other applications and examples available online. Along with an open source, documented and exemplified code, we release reusable containers to allow for convenient and wide usability and increased reproducibility. (10.12688/f1000research.27395.2)
DOI : 10.12688/f1000research.27395.2
Drivers of i-DNA Formation in a Variety of Environments Revealed by Four-Dimensional UV Melting and Annealing
- Cheng Mingpan
- Chen Jielin
- Ju Huangxian
- Zhou Jun
- Mergny Jean-Louis
Journal of the American Chemical Society, American Chemical Society , 2021, 143, pp.7792-7807 . i-DNA is a four-stranded, pH-sensitive structure formed by cytosine-rich DNA sequences. Previous reports have addressed the conditions for formation of this motif in DNA in vitro and validated its existence in human cells. Unfortunately, these in vitro studies have often been performed under different experimental conditions, making comparisons difficult. To overcome this, we developed a four-dimensional UV melting and annealing (4DUVMA) approach to analyze i-DNA formation under a variety of conditions (e.g., pH, temperature, salt, crowding). Analysis of 25 sequences provided a global understanding of i-DNA formation under disparate conditions, which should ultimately allow the design of accurate prediction tools. For example, we found reliable linear correlations between the mid-point of pH transition and temperature (-0.04 ± 0.003 pH unit per 1.0 °C temperature increment) and between the melting temperature and pH (-23.8 ± 1.1 °C per pH unit increment). In addition, by analyzing the hysteresis between denaturing and renaturing profiles in both pH and thermal transitions, we found that loop length, nature of the C-tracts, pH, temperature, and crowding agents all play roles in i-DNA folding kinetics. Interestingly, our data indicate which conformer is more favorable for the sequences with an odd number of cytosine base pairs. Then the h m l pH l f " "-DNAs from human promoter genes were measured under near physiological conditions (pH 7.0, 37 °C). The 4DUVMA method can become a universal resource to analysis the properties of any i-DNA-prone sequence. (10.1021/jacs.1c02209)
DOI : 10.1021/jacs.1c02209
Fluorescent iron‑sulfur centers: Photochemistry of the PetA Rieske protein from Aquifex aeolicus
- Vos Marten H.
- Salman Mayla
- Ramodiharilafy Rivo
- Liebl Ursula
Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier , 2021, 1862 (5), pp.148385 . Cytochrome bc1 complexes are energy-transducing enzymes and key components of respiratory electron chains. They contain Rieske 2Fe-2S proteins that absorb very weakly in the visible absorption region compared to the heme cofactors of the cytochromes, but are known to yield photoproducts. Here, the photoreactions of isolated Rieske proteins from the hyperthermophilic bacterium Aquifex aeolicus are studied in two redox states using ultrafast transient fluorescence and absorption spectroscopy. We provide evidence, for the first time in iron-sulfur proteins, of very weak fluorescence of the excited state, in the oxidized as well as the reduced state. The excited states of the oxidized and reduced forms decay in 1.5 ps and 30 picoseconds, respectively. In both cases they give rise to product states with lifetimes beyond 1 nanosecond, reflecting photo-reduction of oxidized centers as well as photo-oxidation of reduced centers. Potential reaction partners are discussed and studied using site-directed mutagenesis. For the reduced state, a nearby disulfide bridge is suggested as an electron acceptor. The resulting photoproducts in either state may play a role in photoactivation processes. (10.1016/j.bbabio.2021.148385)
DOI : 10.1016/j.bbabio.2021.148385
Mechanism and dynamics of fatty acid photodecarboxylase
- Sorigué Damien
- Hadjidemetriou Kyprianos
- Blangy S.
- Gotthard G.
- Bonvalet A.
- Coquelle Nicolas
- Samire P.
- Aleksandrov Alexey
- Antonucci L.
- Benachir A.
- Boutet S.
- Byrdin Martin
- Cammarata Marco
- Carbajo S.
- Cuine Stephan
- Doak R.
- Foucar L.
- Gorel A.
- Grünbein M.
- Hartmann E.
- Hienerwadel Rainer
- Hilpert M.
- Kloos M.
- Lane T.
- Légeret B.
- Legrand P.
- Li-Beisson Y.
- Moulin S.
- Nurizzo D.
- Peltier G.
- Schirò Giorgio
- Shoeman R.
- Sliwa M.
- Solinas X.
- Zhuang B.
- Barends T.
- Colletier Jacques-Philippe
- Joffre M.
- Royant Antoine
- Berthomieu C.
- Weik Martin
- Domratcheva T.
- Brettel K.
- Vos Marten H.
- Schlichting I.
- Arnoux Pascal
- Müller P.
- Beisson F.
Science, American Association for the Advancement of Science (AAAS) , 2021, 372 (6538), pp.eabd5687 . Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer. (10.1126/science.abd5687)
DOI : 10.1126/science.abd5687