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. 2010 Jun 1;66(Pt 6):665-9.
doi: 10.1107/S1744309110012352. Epub 2010 May 26.

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

Isabelle Petit-Haertlein  1 , Matthew P Blakeley, Eduardo Howard, Isabelle Hazemann, Andre Mitschler, Alberto Podjarny, Michael Haertlein
Affiliations

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

Isabelle Petit-Haertlein et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .

Abstract

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).

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Figures

Figure 1
Figure 1
Amino-acid-specific methyl protonation. (a) Schematic illustration of the major metabolic pathways involved in amino-acid biosynthesis in E. coli. (b) Residue-specific methyl protonation of valine and leucine via the precursor α-ketoisovalerate.
Figure 2
Figure 2
Expression and labelling of AFP. (a) 12% SDS–Tris-tricine polyacrylamide gel electrophoresis. The standard protein bands (kDa) of Precision Plus pre-stained molecular-weight standards (Bio-Rad) are indicated in lane 1. 25 µg purified AFP D VMP + LMP is shown in lane 2. (b) Primary sequence of AFP. Methyl-protonated valine residues are shown in red and leucine residues in blue.
Figure 3
Figure 3
MALDI mass spectrum of AFP. (a) MALDI mass spectrum of AFP D prepared in H2O. (b) MALDI mass spectra of AFP D VMP + LMP prepared in H2O. The mass difference (94 Da) is very close to the expected value of 96 for D–H exchange on 32 methyl groups of valine or leucine residues.
Figure 4
Figure 4
Picture of an AFP D VMP + LMP crystal taken under polarized light. The protein crystallizes in space group P212121, with unit-cell parameters a = 32.7, b = 39.1, c = 46.5 Å at 293 K.
Figure 5
Figure 5
Neutron Laue diffraction pattern. Left: a neutron Laue diffraction pattern from the 0.23 mm3 crystal of AFP D VMP + LMP collected on the LADI-III beamline at the Institut Laue–Langevin. Right: close up of a high-resolution region.
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