Biomedical Engineering Reference
In-Depth Information
Several NMR experimental schemes have been developed recently to address
issues of fast transverse relaxation. In large-molecular-weight proteins in
particular, faster transverse relaxation mainly originates from enhanced
1
H-
1
H
dipolar interactions due to the slower tumbling times. This can be diminished
by expressing proteins in deuterated media to exchange non-labile protons
with deuterium.
1,2
Since
2
H has a lower gyromagnetic (c) ratio than
1
H,
transverse relaxation via dipole-dipole (DD) interaction is strongly suppressed
in deuterated proteins. In addition, the TROSY scheme can productively use
cross-correlation between DD and chemical shift anisotropy (CSA) mechan-
isms and selectively use slowly relaxing spin populations allowing the efficient
detection of backbone NH signals,
3,4
as well as aromatic CH groups.
5
While
the mechanism for the attenuated relaxation is different, i.e., interference
between auto/cross DD relaxation rather than the cross-talk between DD/
CSA, TROSY detection of methyl groups has been also established.
6
TROSY
spectroscopy was used to obtain structural and dynamic information for
systems up to 1 MDa.
7,8
In addition to coherence selection, elaborated site-specific isotope labelling
also helps to reduce unwanted coherence losses. This effect is clearly seen in the
combination of the TROSY scheme with deuteration. In the HN TROSY
experiment, optimal line narrowing of H
N
can be seen in perdeuterated systems
where the DD interaction from directly bonded nitrogens is dominant
compared to that from remote protons.
4
The isolation of observed nuclei is
also the key to the methyl TROSY effect,
6
and was established by selective
methyl labelling using suitable precursors.
9-15
As additional benefit, site-
specific isotope labelling simplifies over-crowded NMR spectra by diminishing
less important information. The stereo-array isotope labelling (SAIL) strategy
is
a
great
example
of
successfully
balancing
quality
against
quantity
of
information content in NMR spectra.
16
An alternative approach to cope with fast transverse relaxation is to detect
lower c-ratio nuclei. After the pioneering work by Markley and co-workers
who used
13
C-detection for studies of small diamagnetic proteins,
17-19
low-c
nuclei detection was largely abandoned in solution-state NMR after more
sensitive
1
H-detected heteronuclear experiments were introduced,
20,21
and
could be recorded routinely on commercial NMR spectrometers.
22-24
Without
considering relaxation while magnetisation transfer and the detection period
etc. and the difference in detecting efficiency for each nucleus, the sensitivity of
NMR experiment is proportional to c
ex
c
1
:
5
ob
B
1
:
0
N
0
:
5
where c
ex
and c
ob
are
gyromagnetic ratios of the excited and the observed nuclei, respectively, B
0
is
the strength of magnetic field and N is the number of scans. Thus,
1
H-
excited-
1
H-detected experiments have an intrinsic sensitivity gain of 32
compared to their corresponding
13
C-excited-
13
C-detected counterparts.
Because of this advantage in sensitivity and the fact that most of the relaxation
pathways are effectively averaged out by rapid molecular tumbling in solution,
most solution biomolecular NMR experiments use
1
H as detected nuclei.
1
H gyromagnetic ratio, is actually a 'double-edged sword'
However, the large
