Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein.

Abstract : The highly homologous type III antifreeze protein (AFP) subfamily share the capability to inhibit ice growth at subzero temperatures. Extensive studies by X-ray crystallography have been conducted, mostly on AFPs from polar fishes. Although interactions between a defined flat ice-binding surface and a particular lattice plane of an ice crystal have now been identified, the fine structural features underlying the antifreeze mechanism still remain unclear owing to the intrinsic difficulty in identifying H atoms using X-ray diffraction data alone. Here, successful perdeuteration (i.e. complete deuteration) for neutron crystallographic studies of the North Atlantic ocean pout (Macrozoarces americanus) AFP in Escherichia coli high-density cell cultures is reported. The perdeuterated protein (AFP D) was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Well shaped perdeuterated AFP D crystals have been grown in D(2)O by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed that with a few exposures of 24 h a very low background and clear small spots up to a resolution of 1.85 A were obtained using a ;radically small' perdeuterated AFP D crystal of dimensions 0.70 x 0.55 x 0.35 mm, corresponding to a volume of 0.13 mm(3).
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Acta crystallographica. Section F, Structural biology communications, John Wiley & Sons Ltd,, 2009, 65 (Pt 4), pp.406-9. 〈10.1107/S1744309109008574〉
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Soumis le : mardi 26 mai 2009 - 17:13:01
Dernière modification le : lundi 19 février 2018 - 14:34:03

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Isabelle Petit-Härtlein, Matthew Blakeley, Eduardo Howard, Isabelle Hazemann, Andre Mitschler, et al.. Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein.. Acta crystallographica. Section F, Structural biology communications, John Wiley & Sons Ltd,, 2009, 65 (Pt 4), pp.406-9. 〈10.1107/S1744309109008574〉. 〈inserm-00384528〉

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