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

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).

Purification and structural analysis of a type III antifreeze protein from the european eelpout Zoarces viviparus.

It has recently been reported that the eelpout Zoarces viviparus synthesizes a family of antifreeze proteins (AFP) similar in sequence to type III AFPs. A method has been set up to separate these antifreeze proteins from blood serum of this teleost species. A total of nine proteins with antifreeze activity have been isolated, several to a purity suited for NMR experiments. One of the proteins, Zvafp13, has been subject to partial structure determination by NMR. 1D- and 2D-H NMR analyses were carried out. In the 1D-experiments it was observed that the protein contained 28 slow-exchanging amides, suggesting a compact structure. The 2D-experiments were utilized to assign observed signals to specific amino acids. From TOCSY- and NOESY-experiments 35 out of a total of 66 amino acids were assigned. The amide exchange pattern, protein primary sequence, chemical shifts and NOE-cross-peaks between amides and -protons in the -sheets suggest that Zvafp13 structurally resembles the recombinant type III AFP rQAE m1.1.

Isolation and some characterisation of antifreeze protein from the European eelpout Zoarces viviparus.

The European eelpout Zoarces viviparus is a common inhabitant in the coastal areas of the eastern Atlantic Ocean and the Baltic region. At least 3 different antifreeze proteins were purified from Z. viviparus serum but more isoforms are most likely present. Two antifreeze proteins with molecular weights of approx. 6.5-7 kDa were characterised and found to share high similarity to the type III antifreeze proteins found in other members of the family Zoarcidae. The antifreeze activity of Z. viviparus antifreeze proteins is concentration dependent and showed a saturation effect when the protein concentration reached 30 mg.ml-1 (crude serum) and 8 mg.ml-1 (partly purified serum) respectively. Further the antifreeze activity was found to be dependent of the buffer osmolality resulting in increasing thermal hysteresis when buffer osmolality was raised from 0 to 1M.

The refined crystal structure of an eel pout type III antifreeze protein RD1 at 0.62-A resolution reveals structural microheterogeneity of protein and solvation.

RD1 is a 7-kDa globular protein from the Antarctic eel pout Lycodichthys dearborni. It belongs to type III of the four types of antifreeze proteins (AFPs) found in marine fishes living at subzero temperatures. For type III AFP, a potential ice-binding flat surface has been identified and is imbedded with side chains capable of making hydrogen bonds with a specific lattice plane on ice. So far, all crystallographic studies on type III AFPs were carried out using the Atlantic ocean pout Macrozoarces americanus as the source organism. Here we present the crystal structure of a type III AFP from a different zoarcid fish, and at an ultra-high resolution of 0.62 A. The protein fold of RD1 comprises a compact globular domain with two internal tandem motifs arranged about a pseudo-dyad symmetry. Each motif of the "pretzel fold" includes four short beta-strands and a 3(10) helix. There is a novel internal cavity of 45 A(3) surrounded by eight conserved nonpolar residues. The model contains several residues with alternate conformations, and a number of split water molecules, probably caused by alternate interactions with the protein molecule. After extensive refinement that includes hydrogen atoms, significant residual electron densities associated with the electrons of peptides and many other bonds could be visualized.

Purification of antifreeze proteins by adsorption to ice.

Antifreeze proteins (AFPs) can protect organisms from freezing injury by adsorbing to ice and inhibiting its growth. We describe here a method where ice, grown on a cold finger, is used to selectively adsorb and purify these ice-binding proteins from a crude mixture. Type III recombinant AFP was enriched approximately 50-fold after one round of partitioning into ice and purified to homogeneity by a second round. This method can also be used to purify non-ice-binding proteins by linkage to AFP domains as demonstrated by the recovery of a 50 kDa maltose-binding protein-AFP fusion from a crude lysate of Escherichia coli.