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Accueil > Publications > Recherche par années > Années 2000 > 2006

2006

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A new NMR solution structure of the SL1 HIV-1(Lai) loop-loop dimer

Dimerization of genomic RNA is directly related with the event of encapsidation and maturation of the virion. The initiating sequence of the dimerization is a short autocomplementary region in the hairpin loop SL1. We describe here a new solution structure of the RNA dimerization initiation site (DIS) of HIV-1(Lai). NMR pulsed field-gradient spin-echo techniques and multidimensional heteronuclear NMR spectroscopy indicate that this structure is formed by two hairpins linked by six Watson-Crick GC base pairs. Hinges between the stems and the loops are stabilized by intra and intermolecular interactions involving the A8, A9 and A16 adenines. The coaxial alignment of the three A-type helices present in the structure is supported by previous crystallography analysis but the A8 and A9 adenines are found in a bulged in position. These data suggest the existence of an equilibrium between bulged in and bulged out conformations in solution.

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Accessibility of tobacco lipid transfer protein cavity revealed by N-15 NMR relaxation studies and molecular dynamics simulations

Plant LTP1 are small helical proteins stabilized by four disulfide bridges and are characterized by the presence of an internal cavity, in which various hydrophobic ligands can be inserted. Recently, we have determined the solution structure of the recombinant tobacco LTP1_1. Unexpectedly, despite a global fold very similar to the structures already known for cereal seed LTP1, its binding properties are different : Tobacco LTP1_1 is able to bind only one monoacylated lipid, whereas cereal LTP1 can bind either one or two. The 3D structure of tobacco LTP1_1 revealed the presence of a hydrophobic cluster, not observed on cereal LTP1 structures, which may hinder one of the two entrances of the cavity defined for wheat LTP1.

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An archaeal peptidase assembles into two different quaternary structures - A tetrahedron and a giant octahedron

Cellular proteolysis involves large oligomeric peptidases that play key roles in the regulation of many cellular processes. The cobalt-activated peptidase TET1 from the hyperthermophilic Archaea Pyrococcus horikoshii (PhTET1) was found to assemble as a 12-subunit tetrahedron and as a 24-subunit octahedral particle. Both quaternary structures were solved by combining x-ray crystallography and cryoelectron microscopy data.

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Automatic first-order multiplet analysis in liquid-state NMR.

The extraction of coupling constant values from NMR spectra is an important step in assigning the configuration of organic molecules. A method for the automatic multiplet analysis in weakly coupled spin systems is described here. It relies on a multi-step procedure that forms the AUJ (AUtomatic J) algorithm. Tolerance to low-magnitude second-order effects is achieved by an efficient multiplet centering and symmetrization step. Time-domain signal analysis through a linear model produces a raw evaluation of coupling constants and their associated multiplicities. The final result is obtained through numerical optimization of the multiplet parameters. Two examples are presented, one from an experimental spectrum, and the other one, of higher complexity, from a computer-simulated multiplet.

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Characterization of the interaction of hypericin with protein kinase C in U-87 MG human glioma cells

A fluorescence imaging technique was used to monitor intracellular localization of protein kinase C (PKC) in U-87 MG human glioma cells in the presence of hypericin (Hyp) and phorbol 12-myristate-13-acetate (PMA). It is shown that PKC localization, which reflects its activity, is influenced by Hyp and this influence is different from that observed for PMA which acts as PKC activator. Fluorescence binding experiments were used to determine the binding constants of Hyp to several isoforms of PKC. The obtained values of K(d)s (similar to 100 nM) suggest that Hyp binds with high affinity to PKC.

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Chemoselective formation of successive triazole linkages in one pot : " Click-click" chemistry

A methodology for the successive regiospecific "clicking" together of three components in one pot via two triazole linkages is reported. The protocol utilizes copper(I)-mediated alkyne-azide cycloaddition reactions combined with a silver(I)-catalyzed TMS-alkyne deprotection under mild hydroalcoholic conditions. We exemplify the approach with peptide-based components to illustrate its compatibility with polyfunctionalized biomolecules. The method constitutes a promising tool for peptide ligation. We also provide a procedure for directly using TMS-alkynes as the cycloaddition partner in classical "click" chemistry.

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