title: 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).
authors.affiliation: ILL-EMBL Deuteration Laboratory, Partnership for Structural Biology, Grenoble, France. haertlein@ill.eu
authors.forename: Isabelle
authors.initials: I
authors.lastname: Petit-Haertlein
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Blakeley
authors.forename: Eduardo
authors.initials: E
authors.lastname: Howard
authors.forename: Isabelle
authors.initials: I
authors.lastname: Hazemann
authors.forename: Andre
authors.initials: A
authors.lastname: Mitschler
authors.forename: Michael
authors.initials: M
authors.lastname: Haertlein
authors.forename: Alberto
authors.initials: A
authors.lastname: Podjarny