Isotope-Labelling of Methyl Groups for NMR Studies of Large Proteins - Recent Developments in Biomolecular NMR - page 9

the supplemented alanine. This approach boosts protein expression levels due
to the use of rich expression medium but, due to the use of rich media, the
incorporation level of [ 13 C]alanine is severely reduced. Scrambling of alanine
can also be effectively eliminated by supplementing minimal expression media
with specific deuterated metabolites (such as isoleucine and a-ketoisovalerate)
that saturate and inhibit metabolic pathways that the supplemented alanine
would leak into. 28 This strategy allows near complete incorporation of labelled
alanine in [ 2 H]-based M9 medium with detectable isotopic scrambling reduced
to less than 1 %.

1.2.2.2 Methionine

The methyl group of methionine is a useful NMR probe as it is isolated at the
end of a long amino acid side-chain. Furthermore, methionine methyl groups
tend to resonate in a largely unpopulated region of the ( 1 H, 13 C) correlation
spectrum (Figure 1.3). The absence of a scalar-coupled carbon means that the
methionine methyl group is a useful probe of protein dynamics.
Isotope-labelled methionine can be added directly to the expression medium.
Unlike alanine, methionine is located at the end of a metabolic pathway and is
therefore not subject to high levels of metabolic scrambling. In a study of the 204
kDa SecA protein (Figure 1.1), Gelis and colleagues added 250 mg L 21 fully
protonated, [e- 13 C]-labelled methionine and observed no scrambling of the
isotope-labelled methyl group. 29 Many isotopic variations of methionine can be
purchased from isotope suppliers or alternatively synthesised using published
protocols. 30,31,32 Fischer and colleagues reported a scheme for the production of
2-oxo-methionine (IUPAC name, 4-methylsulfanyl-2-oxobutanoic acid; CAS
number, 583-92-6), a metabolic precursor of methionine that lacks the a-amino
group. 30 E. coli efficiently assimilate this molecule from the expression solution
and use transaminase enzymes to convert it into methionine.
A recent alternative approach to methionine labelling involves a chemical
post-translational modification of cysteine residues using [ 13 C]-methyl-metha-
nethiosulfonate (MMTS; IUPAC name, methylsulfonylsulfanylmethane; CAS
number, 33784-54-2). 33 MMTS reacts with free cysteine residues to produce (S)-
methylthiocysteine. Thus, MMTS introduces a [ 13 C]-labelled pseudo-methio-
nine methyl group into a target protein, either via an existing cysteine or through
the introduction of suitable sites by site-directed mutagenesis. Methionines and
cysteines are not common in proteins which suggests that using MMTS would
be an attractive way to introduce NMR-visible probes to an uncrowded region
of ( 1 H, 13 C) correlation spectra in a sequence-specific way.

1.2.2.3 Isoleucine
Isoleucine contains two methyl groups. E. coli produce isoleucine by
combining one molecule of pyruvate (IUPAC name, 2-oxopropanoic acid;
CAS number 1892-67-7) and one molecule of a-ketobutyrate (IUPAC name,