Isotope-Labelling of Methyl Groups for NMR Studies of Large Proteins - Recent Developments in Biomolecular NMR - page 10

2-oxo butanoate; CAS number, 600-18-0). Figure 1.4 shows that the two
methyl groups of isoleucine are derived from different sources: carbon-4 of a-
ketobutyrate becomes the d 1 -methyl group and carbon-3 of pyruvate becomes
the c 2 -methyl group. Consequently, different labelling strategies have been
developed that allow isotope-labelling of one or the other.
As with leucine and valine, it is possible to directly supplement the
expression medium with labelled isoleucine. While such an approach works
well, it has in general been supplanted by techniques that utilise biosynthetic
precursors of the final amino acid. 34-36 The precursor-based strategy is
considerably cheaper and allows greater flexibility in the isotopic composition
of the final amino acid.
1.2.2.3.1 Isoleucine d 1 -Methyl Group
The biosynthetic pathway of isoleucine was one of the first to be exploited by
methyl-specific labelling protocols (Figure 1.4). Gardner and Kay demon-
strated that the biosynthetic precursor [3,3- 2 H 2 ,U- 13 C]a-ketobutyrate could be
used to specifically label the d 1 -methyl group of isoleucine without noticeable
isotopic scrambling. 34 The precursor was prepared from [U- 13 C]threonine in
two steps. Initially [U- 13 C]threonine is enzymatically converted into [3- 2 H 2 ,
U- 13 C]a-ketobutyrate using threonine deaminase. The protons at position-3
are exchanged for deuterium by incubation at elevated pH. The final productis
added directly to minimal E. coli expression medium and biosynthetically
converted into isoleucine and used for protein synthesis. Subsequently several
alternative schemes for the synthesis of a-ketobutyrate have been reported 37-39
and, today, many different isotope-labelled varieties are available from isotope
suppliers. 40 Due to the biosynthetic processing of [3- 2 H 2 ,U- 13 C]a-ketobutyrate
it is necessary to use [U- 2 H, U- 13 C]glucose to ensure complete [ 13 C]-labelling of
the isoleucine side-chain and to allow use of NMR experiments that correlate
resonance of the d 1 -methyl group with nuclei in the polypeptide backbone.
Selective protonation of the d 1 -methyl group of isoleucine is now a simple,
robust and inexpensive method that is routinely used for selectively
introducing protonated methyl groups in a perdeuterated protein. The d 1 -
methyl group resonates in an uncrowded region of the 2D ( 1 H, 13 C) correlation
spectra (Figure 1.3). High levels of isotope incorporation (.95 %), without
detectable scrambling, can be achieve by adding 70 mg L 21 a-ketobutyrate to
the expression medium one hour before induction of protein expression. 40
1.2.2.3.2 Isoleucine c 2 -Methyl Group
The c 2 -methyl group of isoleucine is derived from pyruvate (Figure 1.4). Using
[U- 13 C]pyruvate as the sole carbon source in perdeuterated minimal expression
medium produces highly perdeuterated proteins that are partially protonated on
the methyl groups of valine, leucine and isoleucine (c 2 -methyl group only). 41
However, due to metabolic scrambling of the protons at position-3 of pyruvate,