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Caractérisations biochimiques et structurales de la γE cristalline de rat, hydrogénée et perdeutériée en vue d'une étude de diffraction des neutrons.

Abstract : All vertebrate eye lenses are transparent and have a refractive power depending on a smooth refractive index gradient for visible light. This is achieved by the regular arrangement of the fibre cell and by the differential expression of lens specific proteins, the crystallins. The increasing refractive index from the cortex to the nuclear region is associated with increasing concentration of crystallins relative to water. The nuclear region, where the refractive index is the highest, is enriched with γ crystallins, a family of monomeric polypeptides synthesised mainly during the early development, that play a major role in forming the closely packed medium. However, the high concentration is near a critical point whereby very soluble proteins undergo a low energy phase separation driven by the competing forces of protein-water, water-water and protein-protein interactions. This phase separation is crucial in the opacification of lens fibre cells found in certain forms of mammalian cataract. A high resolution neutron diffraction study can supplement the existing X-ray data and allows us to make detailed and thorough analysis of the solvent organisation around the crystalline molecule. This will provide a structural base for studying the effect on protein-water, water-water and protein-protein interactions. This study will be greatly facilitated by the use of fully deuterated protein. Substituting deuterium for hydrogen will reduce the incoherent scattering background, strengthen the high resolution diffraction data and provide an order of magnitude gain in signal to noise. The exchange of the hydrogen by deuterium can be achieved by soaking protein crystals in deuterated liquor but these exchangeable hydrogens represent only 25% of total hydrogens of the protein. The exchange of all hydrogen atoms can only be achieved by an in vivo biosynthesis using a bacterial culture in a deuterated medium (deuterated glycerol and D2O). γE crystalline from rat has been chosen as a model protein system to locate hydrogen atoms of water molecules surrounding the protein and hydrogens involved in different stabilising interactions. Large quantities of the hydrogenated and perdeuterated protein were expressed in Escherichia coli grown in minimal medium and then purified. The level of the isotope substitution on non-exchangeable sites of the protein was found to be 98% by electrospray ionisation mass spectrometry. In the absence of known biochemical activity, the hydrogenated and deuterated γE crystallins were characterised by non denaturing gel electrophoresis, isoelectric point determination and limited proteolysis. There are no major biochemical differences between these two forms of protein. Further characterisations by Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies did not show any significant differences. The hydrogenated and deuterated proteins were crystallised in H2O and D2O buffers. Crystallisation conditions, space groups and cell parameters were found to be the same for all forms of the protein. Comparison of these four forms of γE crystallin revealed no significant structural difference between them at the atomic resolution around 1.4Å. However, the temperature factor variation of the structures at identical resolution (HγEh2o HγEd2o and DγEh2o) depends on the isotope labelling. The structures of the deuterated protein in H2O or the hydrogenated one with D2O solvent have a lower temperature factor. The molecular model of γE crystalline has been obtained at 1.36Å and some new relevant details on the structure and water network surrounding the protein are described in this report. Neutron diffraction data collection cannot be carried out before crystal size improvement, but most importantly, we have shown that perdeuteration itself does not alter the structural features of the protein.
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Contributor : Jean-Baptiste Artero <>
Submitted on : Friday, January 18, 2008 - 3:48:52 PM
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  • HAL Id : tel-00207936, version 1




Jean-Baptiste Artero. Caractérisations biochimiques et structurales de la γE cristalline de rat, hydrogénée et perdeutériée en vue d'une étude de diffraction des neutrons.. Sciences du Vivant [q-bio]. Université Joseph-Fourier - Grenoble I, 2005. Français. ⟨tel-00207936⟩



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