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An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein.

Journal of biomolecular NMR (2008-12-31)
Isabel Ayala, Remy Sounier, Nathalie Usé, Pierre Gans, Jérôme Boisbouvier
ABSTRACT

A strategy for the introduction of ((1)H,(13)C-methyl)-alanine into perdeuterated proteins is described. Specific protonation of alanine methyl groups to a level of 95% can be achieved by overexpressing proteins in M9/D(2)O based bacterial growth medium supplemented with 800 mg/l of 2-[(2)H], 3-[(13)C] L: -alanine. However, though simple, this approach results in undesired, non-specific background labeling due to isotope scrambling via different amino acid metabolic pathways. Following a careful analysis of known metabolic pathways we found that co-addition of perdeuterated forms of alpha-ketoisovalerate-d(7), succinate-d(4) and L: -isoleucine-d(10) with labeled L: -alanine, reduces undesired background labeling to <1%. When combined with recently developed methyl TROSY experiments, this methyl-specific labeling protocol permits the acquisition of excellent quality correlation spectra of alanine methyl groups in high molecular weight proteins. Our cost effective strategy offers a significant enhancement in the level of incorporation of methyl-labeled alanine in overexpressed proteins over previously reported methods.

MATERIALS
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Brand
Product Description

Sigma-Aldrich
Sodium 3-methyl-2-oxobutyrate, 95%