Isotope-Labelling of Methyl Groups for NMR Studies of Large Proteins - Recent Developments in Biomolecular NMR - page 8

Figure 1.4 Metabolic pathways involved in the biogenesis of methyl-group contain-

ing amino acids. This simplified diagram shows only selected inter-
mediates that have been implicated in methyl-labelling protocols. Carbon
atoms have been colour-coded depending on their metabolic source.
Pyruvate (green); a-ketobutyrate and threonine (red); acetyl-CoA (black).
Metabolites have been named according to the original publication
reporting their use. See main text for references, IUPAC names and
Chemical Abstracts Service numbers. Acetolactate is produced from the
condensation of two molecules of pyruvate. The origins of each carbon in
acetolactate and subsequent molecules is denoted by dots.

Specific labelling of the b-methyl group of alanine can be achieved by
supplementing bacterial minimal expression media with 800 mg L 21 of
commercially available [ 13 C]-labelled alanine. 26,28 Different 13 C isotope-
labelling patterns are possible (i.e., [3- 13 C] or [U- 13 C]). [ 13 C]-labelling of both
carbon-2 and carbon-3 (i.e.,C a and C b ) gives access to backbone chemical shift
information. To ensure optimal levels of background deuteration it is
necessary to use [2- 2 H]alanine, which can either be purchased directly or
generated enzymatically from protonated alanine using tryptophan

synthase. 26,28
Alanine is directly synthesised from pyruvate in a reversible transamination
reaction (Figure 1.4). Consequently supplementing a bacterial expression
medium with isotope-labelled alanine will result in severe isotopic scrambling
to other sites in the protein, notably leucine, isoleucine and valine. 28 A
reduction in the level of scrambling has been achieved in a number of ways.
Isaacson and colleagues proposed using [ 13 C]-labelled [2- 2 H]alanine in concert
with a rich perdeuterated expression medium which contains high levels of
deuterated [ 12 C] amino acids. 26 Such a medium also contains deuterated [ 12 C]-
labelled alanine which is incorporated into the target protein at the expense of