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 α‐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 D2O 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 Å from a crystal of dimensions 1.60 × 0.38 × 0.38 mm corresponding to a volume of 0.23 mm3.
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