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Léa Delacour,
Naoko Kotera,
Ténin Traoré,
Sébastien Garcia-Argote,
Céline Puente,
François Leteurtre,
Edmond Gravel,
Nawal Tassali,
Céline Boutin,
Estelle Léonce, Yves Boulard,
Patrick Berthault,
Bernard Rousseau
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ABSTRACT: We describe the synthesis of a highly water-soluble cryptophane 1 that can be seen as a universal platform for the construction of (129) Xe magnetic resonance imaging (MRI)-based biosensors. Compound 1 is easily functionalized by Huisgen cycloaddition and exhibits excellent xenon-encapsulation properties. In addition, 1 is nontoxic at the concentrations typically used for hyperpolarized (129) Xe MRI.
Chemistry 03/2013; · 5.93 Impact Factor
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ABSTRACT: We show that the differentiation between internal and external compartments of various biological cells in suspension can be made via simple NMR spectra of hyperpolarized (129) Xe. The spectral separation between the signals of (129) Xe in these two compartments is already known for red blood cells, because of the strong interaction of the noble gas with hemoglobin. The observation of two separate peaks in the 200-ppm region can be seen with both eukaryotic and prokaryotic cells, some of which are not known to contain paramagnetic proteins in large quantities. Using different experiments in which the cells are lysed, swell or are blocked in G2 phase, we demonstrate that the low-field-shifted peak observed corresponds to xenon in the aqueous pool inside the cells and not in the membranes. The presence of this additional peak is a clear indication of cell integrity, and its integration allows the quantification of the total cell volume. The relaxation time of intracellular xenon is sufficiently long to open up promising perspectives for cell characterization. The exchange time between the inner and outer cell compartments (on the order of 30 ms) renders possible the targeting of intracellular receptors, whereas the observation of chemical shift variations represents a method of revealing the presence of toxic species in the cells.
NMR in Biomedicine 12/2011; 24(10):1264-9. · 3.21 Impact Factor
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ABSTRACT: In a spin: Spin-labeled oligonucleotides produced by click chemistry can be studied by EPR, by using a DEER sequence. This was used to test a complex triple-labeling strategy with damaged DNA. Extensive and accurate analysis of DNA structure and enzymatic repair processes were performed after digestion by EndoIV. Modified DNA structures and DNA-protein interactions can now be readily studied.
ChemBioChem 11/2011; 12(17):2560-3. · 3.94 Impact Factor
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ABSTRACT: The catalytic center of yeast RNA polymerase II and III contains an acidic loop borne by their second largest subunit (Rpb2-(832)GYNQED(837), Rpc128-(764)GYDIED(769)) and highly conserved in all cellular and viral DNA-dependent RNA polymerases. A site-directed mutagenesis of this dicarboxylic motif reveals its strictly essential character in RNA polymerase III, with a slightly less stringent pattern in RNA polymerase II, where rpb2-E836Q and other substitutions completely prevent growth, whereas rpb2-E836A combines a dominant growth defect with severe lethal sectoring. A mild but systematic increase in RNA polymerase occupancy and a strict dependency on the transcript cleavage factor TFIIS (Dst1) also suggest a slower rate of translocation or higher probability of transcriptional stalling in this mutation. A conserved nucleotide triphosphate funnel domain binds the Rpb2-(832)GYNQED(837) loop by an Rpb2-R(1020)/Rpb2-D(837) salt-bridge. Molecular dynamic simulations reveal a second bridge (Rpb1-K(752)/Rpb2-E(836)), which may account for the critical role of the invariant Rpb2-E(836). Rpb2-E(836) and the funnel domain are not found among the RNA-dependent eukaryotic RNA polymerases and may thus represent a specific adaptation to double-stranded DNA templates.
Current Genetics 07/2011; 57(5):327-34. · 2.56 Impact Factor
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Céline Boutin,
Antoine Stopin,
Fatimazohra Lenda,
Thierry Brotin,
Jean-Pierre Dutasta,
Nadège Jamin,
Alain Sanson, Yves Boulard,
François Leteurtre,
Gaspard Huber,
Aurore Bogaert-Buchmann,
Nawal Tassali,
Hervé Desvaux,
Marie Carrière,
Patrick Berthault
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ABSTRACT: For detection of biological events in vitro, sensors using hyperpolarized (129)Xe NMR can become a powerful tool, provided the approach can bridge the gap in sensitivity. Here we propose constructs based on the non-selective grafting of cryptophane precursors on holo-transferrin. This biological system was chosen because there are many receptors on the cell surface, and endocytosis further increases this density. The study of these biosensors with K562 cell suspensions via fluorescence microscopy and (129)Xe NMR indicates a strong interaction, as well as interesting features such as the capacity of xenon to enter the cryptophane even when the biosensor is endocytosed, while keeping a high level of polarization. Despite a lack of specificity for transferrin receptors, undoubtedly due to the hydrophobic character of the cryptophane moiety that attracts the biosensor into the cell membrane, these biosensors allow the first in-cell probing of biological events using hyperpolarized xenon.
Bioorganic & medicinal chemistry 07/2011; 19(13):4135-43. · 2.82 Impact Factor
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ABSTRACT: Cadmium is a heavy metal and a pollutant that can be found in large quantities in the environment from industrial waste. Its toxicity for living organisms could arise from its ability to alter thiol-containing cellular components. Glutathione is an abundant tripeptide (γ-Glu-Cys-Gly) that is described as the first line of defence against cadmium in many cell types. NMR experiments for structure and dynamics determination, molecular simulations, competition reactions for metal chelation by different metabolites (γ-Glu-Cys-Gly, α-Glu-Cys-Gly and γ-Glu-Cys) combined with biochemical and genetics experiments have been performed to propose a full description of bio-inorganic reactions occurring in the early steps of cadmium detoxification processes. Our results give unambiguous information about the spontaneous formation, under physiological conditions, of the Cd(GS)(2) complex, about the nature of ligands involved in cadmium chelation by glutathione, and provide insights on the structures of Cd(GS)(2) complexes in solution at different pH. We also show that γ-Glu-Cys, the precursor of glutathione, forms a stable complex with cadmium, but biological studies of the first steps of cadmium detoxification reveal that this complex does not seem to be relevant for this purpose.
FEBS Journal 10/2010; 277(24):5086-96. · 3.79 Impact Factor
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ABSTRACT: In the (129)Xe NMR-based biosensing approach in which the hyperpolarized noble gas is transported to biological receptors for a sensitive molecular imaging, cryptophanes are excellent xenon host systems. However to avoid formation of self-organized systems, these hydrophobic cage molecules can be rendered water soluble by introduction of ionic groups. We show that the sensitivity of xenon to its local environment and the presence of these ionic functions can lead to interesting properties. For a first water-soluble cryptophane derivative, we show that a precise monitoring of the local pH can be performed. For a second cryptophane, the presence of ionic groups close to the cryptophane cavity modifies the xenon binding constant and in-out exchange rate. The latter allows the tuning of physical properties of xenon-cryptophane interactions without resorting to a change of the cavity size. These results open new perspectives on the influence of chemical modifications of cryptophanes for optimizing the biosensor properties.
Chemistry 09/2010; 16(43):12941-6. · 5.93 Impact Factor
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ABSTRACT: Double electron-electron resonance (DEER) was applied to determine nanometre spin-spin distances on DNA duplexes that contain selected structural alterations. The present approach to evaluate the structural features of DNA damages is thus related to the interspin distance changes, as well as to the flexibility of the overall structure deduced from the distance distribution. A set of site-directed nitroxide-labelled double-stranded DNA fragments containing defined lesions, namely an 8-oxoguanine, an abasic site or abasic site analogues, a nick, a gap and a bulge structure were prepared and then analysed by the DEER spectroscopic technique. New insights into the application of 4-pulse DEER sequence are also provided, in particular with respect to the spin probes' positions and the rigidity of selected systems. The lesion-induced conformational changes observed, which were supported by molecular dynamics studies, confirm the results obtained by other, more conventional, spectroscopic techniques. Thus, the experimental approaches described herein provide an efficient method for probing lesion-induced structural changes of nucleic acids.
Nucleic Acids Research 04/2009; 37(10):3165-76. · 8.03 Impact Factor
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Journal of the American Chemical Society 01/2009; 130(49):16456-7. · 9.91 Impact Factor
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Angewandte Chemie International Edition 02/2008; 47(4):735-7. · 13.45 Impact Factor
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ChemPhysChem 11/2007; 8(14):2082-5. · 3.41 Impact Factor
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ABSTRACT: Rpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large N-terminal helix and a short motif at the end of the module are critical in vivo. Lethal or conditional mutants of the C-terminal globe altered the binding of Rpb5 to Rpb1-beta25/26 (prolonging the Bridge helix) and Rpb1-alpha44/47 (ahead of the Switch 1 loop and binding Rpb5 in a two-hybrid assay). The large intervening segment of Rpb1 is held across the DNA Cleft by Rpb9, consistent with the synergy observed for rpb5 mutants and rpb9Delta or its RNA polymerase I rpa12Delta counterpart. Rpb1-beta25/26, Rpb1-alpha44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-alpha46/47 helix bundle were universally conserved. We conclude that the main function of the dual Rpb5-Rpb1 binding and the Rpb9-Rpb1 interaction is to hold the Bridge helix, the Rpb1-alpha44/47 helix bundle and the Switch 1 loop into a closely packed DNA-binding fold around the transcription bubble, in an organization shared by the two other nuclear RNA polymerases and by the archaeal and viral enzymes.
Nucleic Acids Research 02/2007; 35(2):634-47. · 8.03 Impact Factor
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ABSTRACT: We have carried out a solution study of two non-labelled self-complementary DNA dodecamers d(GACTGTACAGTC)2 and d(GACTGTGCAGTC)2 by NMR, the second sequence composed of two G-T mismatches. Structures were determined using distances extracted from NOE effects alone or using both NOE and RDC constraints, measured in three different liquid crystalline media. We ensured that our data on the influence of the mesogen on the DNA structures, and the way in which the RDCs were incorporated as constraints in the protocol refinement, were consistent. We also tested the influence of different sets of RDCs and the best means of optimizing the calculation of D(a) and R. Resolution and accuracy of the ten best energy final structures were compared. The addition of a small set of RDC constraints significantly improves the final determined structures. We took advantage of the specificity of the RDC, i.e. it contains orientational information, and explored the global shape of the DNA duplexes; it was found that the duplexes do not have a large curvature. For the G-T base pair, we observed, in this particular sequence (tandem of G-T mismatches), a new pattern of base pairing, which involved the formation of a bifurcated hydrogen bond.
Magnetic Resonance in Chemistry 01/2007; 44(12):1081-9. · 1.44 Impact Factor
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ABSTRACT: Yaf9 is one of three proteins in budding yeast containing a YEATS domain. We show that Yaf9 is part of a large complex and that it coprecipitates with three known subunits of the NuA4 histone acetyltransferase. Although Esa1, the catalytic subunit of NuA4, is essential for viability, we found that yaf9 Delta mutants are viable but hypersensitive to microtubule depolymerizing agents and synthetically lethal with two different mutants of the mitotic apparatus. Microtubules depolymerized more readily in the yaf9Delta mutant compared to the wild type in the presence of nocodazole, and recovery of microtubule polymerization and cell division from limiting concentrations of nocodazole was inhibited. Two other NuA4 mutants (esa1-1851 and yng2 Delta) and nonacetylatable histone H4 mutants were also sensitive to benomyl. Furthermore, wild-type budding yeast were more resistant to benomyl when grown in the presence of trichostatin A, a histone deacetylase inhibitor. These results strongly suggest that acetylation of histone H4 by NuA4 is required for the cellular resistance to spindle stress.
Molecular and Cellular Biology 10/2003; 23(17):6086-102. · 5.53 Impact Factor
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ABSTRACT: The solution structures of a trisaccharide and a pentasaccharide containing the Lewis(x) motif were determined by two independent approaches using either dipolar cross-relaxation (NOE) or residual dipolar coupling (RDC) data. For the latter, one-bond 13C[bond](1)H RDC enhanced by two different mineral liquid crystals were used alone. Home-written programs were employed firstly for measuring accurately the coupling constants in the direct dimension of non-decoupled HSQC experiments, secondly for transforming each RDC data set into geometrical restraints. In this second program, the complete molecular structure was expressed in a unique frame where the alignment tensor is diagonal. Assuming that the pyranose rings are rigid, their relative orientation is defined by optimizing the glycosidic torsion angles. For the trisaccharide, a good agreement was observed between the results of both approaches (NOE and RDC). In contrast, for the pentasaccharide, strong discrepancies appeared, which seem to result from interactions between the pentasaccharide and the mesogens, affecting conformational equilibrium. This observation is of importance, as it reveals that using simultaneously NOE and RDC can be hazardous as the former represent 99% of the molecules free in solution, whereas the latter correspond to less than 1% of the structure bound to the mesogen.
Carbohydrate Research 09/2003; 338(17):1771-85. · 2.33 Impact Factor
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ABSTRACT: The N-(2-deoxy-beta3-D-erythro-pentofuranosyl) formamide residue results from a ring fragmentation product of thymine or cytosine. The presence of a formamide-adenine base pair in the sequence 5'd(AGGAACCACG).d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. There are two possible isomers for the formamide side chain, either cis or trans. For each isomer, we observed an equilibrium in solution between two forms. First, a species where the formamide is intrahelical and paired with the facing adenine. For the cis isomer, the formamide is in a syn conformation and two hydrogen bonds with adenine are formed. The trans isomer is in an anti conformation and a single hydrogen bond is observed. In the second form, whatever the isomer, the formamide is rejected outside the helix, whereas the adenine remains inside.
Canadian Journal of Physiology and Pharmacology 08/2002; 80(7):609-17. · 1.95 Impact Factor
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ABSTRACT: Determination of the solution structure of the duplex d(GCAAGTC(HE)AAAACG)*d(CGTTTTAGACTTGC) containing a 3-(2-hydroxyethyl)-2'-deoxyuridine*deoxyadenine (HE*A) base pair is reported. The three-dimensional solution structure, determined starting from 512 models via restrained molecular mechanics using inter-proton distances and torsion angles, converged to two final families of structures. For both families the HE and the opposite A residues are intrahelical and in the anti conformation. The hydroxyethyl chain lies close to the helix axis and for one family the hydroxyl group is above the HE*A plane and in the other case it is below. These two models were used to start molecular dynamic calculations with explicit solvent to explore the hydrogen bonding possibilities of the HE*A base pair. The dynamics calculations converge finally to one model structure in which two hydrogen bonds are formed. The first is formed all the time and is between HEO4 and the amino group of A, and the second, an intermittent one, is between the hydroxyl group and the N1 of A. When this second hydrogen bond is not formed a weak interaction CH...N is possible between HEC7H2 and N1A21. All the best structures show an increase in the C1'-C1' distance relative to a Watson-Crick base pair.
Nucleic Acids Research 04/2002; 30(6):1371-8. · 8.03 Impact Factor
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ABSTRACT: We investigated the behaviour of a 15mer DNA duplex, [5′d(CAGAGTCACTGGCTC)3′]. [5′d(GAGCCAG)3′ + 5′d(GACTCTG)3′] which contained an adenine opposite the gap. Analysis of the NMR data showed the existence of one major species, which was in equilibrium with two minor species. Their relative concentrations varied as a function of pH with a pKa of ≈ 4.5. For the major species, the duplex was globally in B conformation with the central adenine stacked in the helix. The two G·C base pairs adjacent to the central adenine were well formed and a gap was present in front of this adenine. For the minor species, major structural perturbations occurred in the centre of the duplex. At neutral pH, the central adenine was involved in a G·A mismatch with G23 adjacent to the gap. Cytosine C7 was then extrahelical and no gap was observed. Under these conditions, the major neutral species corresponded to 70% of the total and the minor species to 30%. At acidic pH, the central adenine of the minor species was protonated and was involved in a G(syn)·A+(anti) mismatch. The difference is that C9 is now extrahelical and G22 is implicated in the mispair. Three-dimensional models were built to initiate molecular dynamic simulations, which were in good agreement with the NMR data. Their structural stability in terms of hydrogen bonding and their flexibility are discussed and the biological significance for the interaction with DNA polymerase is evoked.
European Journal of Biochemistry. 08/1999; 264(1):120 - 131.
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ABSTRACT: Two mismatches, G·G and I·I, have been incorporated at the central position of 5‘d(GCCACXAGCTC)·d(GAGCTXGTGGC) in order to carry out NMR and molecular dynamics studies. These duplexes constitute the sequence 29−39 of the K-ras gene coding for the glycine 12, a hot spot for mutation. The NMR spectra show that the duplexes are not greatly distorted by the introduction of the mismatches and their global conformation is that of a canonical B-form double helix. For the duplex containing the G·G mismatch, we propose for the major species, a type of pairing involving one hydrogen bond between the imino group of one central guanine and the carbonyl group of the opposite guanine. Both bases are in an anti conformation. Two conformations, with the same donor and acceptor pattern can coexist, one is obtained from the other by a 180° rotation about the pseudodyadic axis. Exchange between the two forms is observed by NMR at low temperature. A minor species involving hydrogen bonding between the guanine amino group and the carbonyl group of the guanine on the opposite strand may also exist as shown by the molecular dynamics calculations. For the I·I mismatch we observe the same major species, i.e., hydrogen bonding between an imino proton of one base and the carbonyl group of the base on the opposite strand with both bases in an anti conformation. Exchange between these two conformations is faster than for the G·G mismatch. Further, we observe that the I·I mismatch adopts a minor conformation, in which one or other of the bases is in the syn conformation.
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