An efficient protocol for the complete incorporation of methyl-protonated alanine in perdeuterated protein.
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.
Ayala
Isabel
I
Institut de Biologie Structurale Jean-Pierre Ebel, UMR 5075, Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, Université Joseph-Fourier, 41 rue Jules Horowitz, 38027, Grenoble Cedex, France.
Sounier
Remy
R
Usé
Nathalie
N
Gans
Pierre
P
Boisbouvier
Jérôme
J
D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D003470:Culture Media; D003903:Deuterium / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D045782:Hemiterpenes; D006801:Humans; D007532:Isoleucine / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D007651:Keto Acids / Q000737:chemistry / Q000378:metabolism; D008292:Malate Synthase / Q000737:chemistry / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019802:Succinic Acid / Q000737:chemistry / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002247:Carbon Isotopes; D003470:Culture Media; D045782:Hemiterpenes; D007651:Keto Acids; D011506:Proteins; D025801:Ubiquitin; D007532:Isoleucine; C001505:alpha-ketoisovalerate; D019802:Succinic Acid; D003903:Deuterium; D008292:Malate Synthase; D000409:Alanine