Biomedical Engineering Reference
In-Depth Information
12.6 NMR Method Development
Many NMR studies of membrane proteins rely on approaches to structure
determination adapted from water-soluble proteins involving backbone
assignment using TROSY-based spectra, followed by structure determination
using NOE and other restraints discussed in the previous sections, which can
yield extremely high-quality structures.
19
At the same time, a number of
alternative approaches for data acquisition and processing are emerging which
may facilitate NMR studies of large membrane proteins.
12.6.1
13
C Direct Detection
Whilst
13
C has a four-fold lower gyromagnetic ratio than
1
H resulting in a
lower sensitivity, improved cryoprobe technology has started to make carbon-
observation experiments more competitive. The Boltzman-related loss in
sensitivity is partly compensated by the increased T
2
values of
13
C when
compared with protons, resulting in spectra with sharper lines. In the crowded
spectra typical of membrane proteins, this resolution enhancement can be a
significant advantage which can be further improved through side-chain
deuteration.
173
Carbon detection leads to experiments with shorter magnetisa-
tion transfers when compared with out-and-back type proton acquisition
experiments used, for example, for backbone assignment, therefore reducing
the loss due to coherence transfer. Furthermore, in contrast to
1
H,
15
N spectra,
intensity losses due to solvent suppression artefacts and exchange of NH
groups are also avoided. Many carbon-detected pulse sequences for backbone
assignment, measurement of RDCs, dihedral angle restraints and cross-
correlated relaxation are already available.
173,241-246
Schemes to overcome
problems due to the effects of the large homonuclear one-bond
13
C scalar
couplings during detection have been developed, for example for the
13
CO-
13
C
a
coupling.
247-250
However,
13
CO nuclei suffer from faster relaxation
due to the large chemical shift anisotropy (CSA), which causes particular
problems for large, slow-tumbling systems. Direct detection on C
a
is beneficial
due to the lower CSA
242
but is complicated due to the possibility of coupling to
both the carbonyl and C
b
. Alternate
13
C-
12
C labelling has been proposed in
order to overcome the large
13
C-
13
C coupling thus permitting detection on the
slower relaxing
13
C
a
nucleus.
251
Simultaneous detection of both
13
C
a
and N
sequential connectivities via 3D CA(N)CA experiments, provides a particularly
robust assignment strategy.
252
In combination with such an alternate labelling
scheme, direct correlations between
13
C nuclei in neighbouring amino acids are
possible using the weak
3
J coupling via a CACA-TOCSY experiment, yielding
improved sensitivity over CA(N)CA experiments; dihedral angle information
can also be obtained along with side-chain assignments for some amino
acids.
253
Simulations of slower tumbling systems demonstrate the potential of
such experiments for large proteins.
