Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studies

PETIT-HAERTLEIN, ISABELLE; BLAKELEY, MATTHEW P.; HOWARD, EDUARDO I; HAZEMANN, ISABELLE; MITSCHLER, ANDRE; PODJARNY, ALBERTO; HAERTLEIN, MICHAEL

ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS

WILEY-BLACKWELL PUBLISHING, INC

Año: 2010

1744-3091

Antifreeze proteins (AFPs) are found in different species from polar, alpine andsubarctic regions, where they serve to inhibit ice-crystal growth by adsorption toice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus)AFP has been used as a model protein to develop protocols for aminoacid-specific hydrogen reverse-labelling of methyl groups in leucine and valineresidues using Escherichia coli high-density cell cultures supplemented with theamino-acid precursor alpha-ketoisovalerate. Here, the successful methyl protonation(methyl reverse-labelling) of leucine and valine residues in AFP is reported.Methyl-protonated AFP was expressed in inclusion bodies, refolded indeuterated buffer and purified by cation-exchange chromatography. Crystalswere grown in D2O buffer by the sitting-drop method. Preliminary neutron Lauediffraction at 293 K using LADI-III at ILL showed in a few 24 h exposures avery low background and clear small spots up to a resolution of 1.80 A ˚ from acrystal of dimensions 1.60 x 0.38 x 0.38 mm corresponding to a volume of0.23 mm3.