Perdeuteration, purification, crystallization and preliminary neutron diffraction of an ocean pout type III antifreeze protein. - Archive ouverte HAL Access content directly
Journal Articles Acta crystallographica Section F : Structural biology communications [2014-...] Year : 2009

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

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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).

Dates and versions

inserm-00384528 , version 1 (26-05-2009)

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Isabelle Petit-Härtlein, Matthew P. 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 [2014-..], 2009, 65 (Pt 4), pp.406-9. ⟨10.1107/S1744309109008574⟩. ⟨inserm-00384528⟩
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