Kinetic study of the self-assembly of brome mosaic virus capsid

The protein of brome mosaic virus can self assemble in-vitro to form empty capsids. In the absence of RNA at pH=7 and 0.5 M KCl there is a dynamic equilibrium between monomers and oligomers. At pH=5 the protein assembles into empty capsids. The kinetics of this assembly, triggered by pH jump from neutral to acidic pH, was investigated by X-ray and light scattering.Cryoelectron microscopy observations suggested that reconstitution is achieved by progressive incorporation of small building units in a spherical shell. This hypothesis has been tested by the analysis of the scattering data in terms of four classes of incomplete capsids represented as spherical shells with holes of different sizes. The time dependence of the population of each class was determined by a least squares analysis of the experimental data. Although the basic polymerizing unit has not been uniquely characterized, the results are compatible with a dimer for this species. The characteristic times for capsid assembly are found to vary as the inverse of the square of the concentration

An apparatus for stopped-flow X-ray scattering

A stopped-flow apparatus and control system, designed for the study of rapid reaction kinetics in solution by X-ray scattering, is described. Inspired from a commercial stopped-flow unit used with UV and visible light, the X-ray device has a dead-time of 80 ms. Results are presented for the polymerization of the coat protein of Brome mosaic virus following a pH jump, using a small angle X-ray scattering instrument at Hasylab (Hamburg).Cet article décrit un appareil de « stopped-flow » et son système de contrôle destiné à l'étude, par diffusion des rayons X, de cinétiques rapides de réactions en solution. Inspiré d'un appareil commercial utilisant les UV et la lumière visible, l'appareil à rayons X a un temps mort de 80 ms. Des résultats sont présentés pour la polymérisation de la protéine de conque du virus de la mosaïque du Brome par saut de pH, en utilisant l'installation de diffusion de rayons X à petits angles de Hasylab (Hambourg)

An apparatus for stopped-flow X-ray scattering

A stopped-flow apparatus and control system, designed for the study of rapid reaction kinetics in solution by X-ray scattering, is described. Inspired from a commercial stopped-flow unit used with UV and visible light, the X-ray device has a dead-time of 80 ms. Results are presented for the polymerization of the coat protein of Brome mosaic virus following a pH jump, using a small angle X-ray scattering instrument at Hasylab (Hamburg)

A Sulfur Tripod Glycoconjugate that Releases a High-Affinity Copper Chelator in Hepatocytes

International audienceReleased in the cell: Three N-acetylgalactosamine units, which recognize the asialoglycoprotein receptor, were tethered through disulfide bonds to the three coordinating thiol functions of a sulfur tripod ligand that has a high affinity for CuI (see scheme). The resulting glycoconjugate can be considered as a prodrug, because after uptake by hepatic cells the intracellular reducing glutathione (GSH) releases the high-affinity intracellular CuI chelator

Hepatocyte Targeting and Intracellular Copper Chelation by a Thiol-Containing Glycocyclopeptide

International audienceMetal overload plays an important role in several diseases or intoxications, like in Wilson’s disease, a major genetic disorder of copper metabolism in humans. To efficiently and selectively decrease copper concentration in the liver that is highly damaged, chelators should be targeted at the hepatocytes. In the present work, we synthesized a molecule able to both lower intracellular copper, namely Cu(I), and target hepatocytes, combining within the same structure a chelating unit and a carbohydrate recognition element. A cyclodecapeptide scaffold displaying a controlled conformation with two independent faces was chosen to introduce both units. One face displays a cluster of carbohydrates to ensure an efficient recognition of the asialoglycoprotein receptors, expressed on the surface of hepatocytes. The second face is devoted to metal ion complexation thanks to the thiolate functions of two cysteine side-chains. To obtain a chelator that is active only once inside the cells, the two thiol functions were oxidized in a disulfide bridge to afford the glycopeptide P3. Two simple cyclodecapeptides modeling the reduced and complexing form of P3 in cells proved a high affinity for Cu(I) and a high selectivity with respect to Zn(II). As expected, P3 becomes an efficient Cu(I) chelator in the presence of glutathione that mimics the intracellular reducing environment. Finally, cellular uptake and ability to lower intracellular copper were demonstrated in hepatic cell lines, in particular in WIF-B9, making P3 a good candidate to fight copper overload in the liver