Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studies.
Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-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 in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D(2)O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed in a few 24 h exposures a very low background and clear small spots up to a resolution of 1.80 A from a crystal of dimensions 1.60 x 0.38 x 0.38 mm corresponding to a volume of 0.23 mm(3).
ILL-EMBL Deuteration Laboratory, Partnership for Structural Biology, 6 Rue Jules Horowitz, 38042 Grenoble, France.
Isabelle
I
Petit-Haertlein
Matthew P
MP
Blakeley
Eduardo
E
Howard
Isabelle
I
Hazemann
Andre
A
Mitschler
Alberto
A
Podjarny
Michael
M
Haertlein
D000595:Amino Acid Sequence; D000818:Animals; D021343:Antifreeze Proteins, Type III / Q000737:chemistry* / Q000235:genetics; D003460:Crystallization; D005399:Fishes; D015870:Gene Expression; D007930:Leucine / Q000737:chemistry; D008745:Methylation; D008969:Molecular Sequence Data; D033363:Neutron Diffraction; D011522:Protons; D014633:Valine / Q000737:chemistry
D021343:Antifreeze Proteins, Type III; D011522:Protons; D007930:Leucine; D014633:Valine