Isotope-Labelling of Methyl Groups for NMR Studies of Large Proteins - Recent Developments in Biomolecular NMR - page 4

The major source of relaxation for 1 H nuclei in higher molecular weight
proteins is the large number of dipolar interactions with neighbouring protons.
For most heteronuclei ( 15 Nor 13 C), however, the dominant factor is the direct
dipolar interaction(s) with covalently bound proton(s). To overcome this
limitation, proteins can be expressed in perdeuterated expression medium.
Protons are then re-introduced at labile sites (e.g., HN) by purifying (or, if
necessary, refolding) the protein in H 2 O-based buffers. This approach ensures
that backbone-directed NMR experiments that utilise the amide proton are
still applicable. Perdeuteration reduces proton density by introducing
deuterium at all non-labile sites and therefore reduces the transverse relaxation
rates of the remaining protons. The consequent narrowing of 1 H signal
linewidths can make a dramatic difference to NMR spectra of larger proteins.
Furthermore, deuteration of aliphatic [ 13 C] sites considerably extends the
lifetime of transverse coherences which is critical when applying three-
dimensional (3D) or four-dimensional (4D) NMR experiments to proteins
larger than 20-30 kDa.
Using a [U- 2 H, 13 C, 15 N]-labelled amide-reprotonated sample it is possible to
obtain backbone resonance assignments for proteins and protein complexesup
to 100-150 kDa. To date the largest single chain protein for which near
complete backbone resonance assignments have been acquired is the 723
residue, 82 kDa, bacterial enzyme, malate synthase G (MSG; ref. 8;
Figure 1.1). Backbone resonance assignments of larger systems have been
determined, but only in cases where the protein target exists as a homo-
oligomer (e.g., refs. 9 and 10).
1.1.3 NMR Experiments Designed for Larger Systems
By isolating each proton from other protons of the protein, a high level of
deuteration is an efficient way to narrow the linewidths of the remaining 1 H
spins. Nevertheless, 1 H/ 2 H substitution has only a moderate effect on the
NMR signal of heteronuclei ( 15 Nor 13 C) that are directly bonded to the
remaining 1 H spins. As the acquisition of high-quality 2D ( 1 H, 15 N) or ( 1 H,
13 C) NMR spectra is a prerequisite for the NMR study of a large protein,
considerable effort has been spent during the last 15 years to develop new
NMR tools that optimise the relaxation of the NMR signals of both 1 Hand
covalently bonded 15 Nor 13 C spins. This concept is known as Transverse
Relaxation Optimised SpectroscopY (TROSY). 11,12 In an isolated two-spin
system involving covalently bonded nuclei, e.g., a 1 H- 15 Nor 1 H- 13 C pair, the
main spin interactions are dipolar interactions between nuclei and the chemical
shift anisotropy (CSA) of each spin. As the same molecular motions modulate
these interactions they can give rise to interference effects. Such effects, also
called cross-correlated relaxation, modulate the relaxation of the different
NMR observable transitions. 13 The so-called TROSY experiments enhance
resolution and sensitivity of NMR experiments of large biomolecules by
selecting transitions(s) with more favourable relaxation properties. Since the