Biomedical Engineering Reference
In-Depth Information
signal.
21
In much larger perdeuterated proteins the rapidly relaxing component
disappears during the course of the pulse sequence leaving only the slowly
relaxing component to be detected. 2D (
1
H,
13
C) HMQC (also called methyl-
TROSY) spectra of large perdeuterated selectively methyl-protonated proteins
show a high level of sensitivity and signal resolution (Figure 1.2). In recent years,
the combination of methyl-TROSY spectroscopy with residue-type-specific
methyl labelling has allowed solution NMR studies of very large protein systems
of several hundreds of kDa. The aim of this chapter is to present an overview of
recently developed isotopic-labelling methods that allow such large biomole-
cular systems to be investigated by NMR spectroscopy.
1.2
Using Methyl Groups as Probes for NMR
Spectroscopy
1.2.1 Why the Methyl Group?
As molecular size increases it becomes increasingly difficult to rely on NH-
based NMR spectra. If [U-
2
H,
13
C]glucose has been used as the sole carbon
source, a perdeuterated labile site-reprotonated (e.g., all NH, OH, etc.) protein
will still have a protonation level around 20 %. This level of protonation
becomes detrimental for proteins larger than 100 kDa. For such proteins, the
methyl group has become the NMR probe of choice. Each methyl group
comprises three protons which rotate rapidly around the methyl symmetry
axis. The consequent three-fold degeneracy of the chemical shifts of the methyl
protons greatly increases sensitivity compared to the backbone amide proton.
Furthermore, methyl groups are often located at the end of long amino acid
side-chains and are generally more dynamic than backbone amide protons.
In general, methyl groups resonate in a largely uncrowded region of the 2D
(
1
H,
13
C) spectrum (Figure 1.3). Methyl-group-containing residues are usually
common and well dispersed in the amino acid sequence and are present both in
the hydrophobic core of proteins and at interaction sites. Using methyl-
TROSY NMR experiments it is possible to acquire high-quality NMR spectra
of methyl-protonated perdeuterated large proteins,
22
potentially in as little
time as 1 s (ref. 23). Thus, methyl groups are excellent probes of protein
structure and dynamics, particularly for very large proteins.
1.2.2 Strategies for Selective Protonation of Methyl Groups in
Perdeuterated Proteins
There are six methyl-containing amino acids found in proteins, excluding post-
translational modifications. Over the past 15 years a variety of strategies for
selective labelling of methyl groups in proteins have been proposed. The
objective of these labelling approaches is to produce highly deuterated (i.e.,
.98 %) proteins with targeted [
13
CH
3
]-labelling at residue-specific methyl sites.
Carbon-labelling
patterns
in
the
rest of
the
side-chain
can
vary
and
be
