Biomedical Engineering Reference
In-Depth Information
for fast-relaxing systems. While, it provides a large Zeeman polarisation at the
beginning of an experiment and induces a larger signal in the receiver coil, the
stronger dipolar interaction also causes a faster decay during the pulse
sequences and detection. Since the peak heights are proportional to the integral
over the detected free induction decay (FID) and not only to the initial
amplitude, a slowly decaying signal of lower initial FID amplitude may havea
superior signal-to-noise ratio (S/N) over a rapidly relaxing signal of higher
initial FID amplitude. Moreover, broad linewidths of the detected signals may
lead to poor resolution of the resulting spectrum and severe signal overlap.
Thus, it is reasonable to consider direct detection of low-c nuclei in fast
relaxation systems. Indeed, the recent arrival of cryogenic probes optimised for
13
C detection has already led to a revival of direct detection experiments.
25-29
Similar to the sensitivity gain obtained in cryogenic probes optimised for
1
H-
detection, significant sensitivity gains were observed with probes optimised for
13
C- and
15
N-detection when the cold X-coil was placed inside and pre-
amplifiers were also cold.
30
A series of
13
C-detected 'proton-less' NMR
experiments developed by Bertini's group are a hallmark of this field as they
provide a set of
13
C-detected (mainly C9) experiments which enable the
characterisation and sequence-specific assignment of moderate-size proteins.
27
These experimental schemes have been successfully applied to paramagnetic
systems, which exhibit very fast relaxation of the spins near the paramagnetic
centres where
1
H-detection is often impossible.
Other cases where heteronuclear detection might help include systems with
unfavourable dispersion in
1
H resonances. Typical examples include unfolded
proteins,
31
DNA/RNA systems,
32-34
as well as sugar chains.
35
Applications
include studies of an unfolded protein in living cells.
36
In these systems the
dispersion of
1
H resonances is rather poor and becomes a major hindrance
even for moderate-size polypeptides. Another use is for detecting peptide
signals for which the amide protons exchange too fast with solvent to be
observed, such as exposed amides above pH 8 or at higher temperatures.
37
Signals may also be exchange broadened due to multiple conformations, and
the broadening effect is more severe for protons than for low-c nuclei.
Interestingly, exchange broadening is often associated with functional
importance, such as found in the catalytic centres of enzymes and/or in
interaction sites. By using
13
C-detected experiments, correlations between non-
labile nuclei of a given biomolecule can be obtained with high resolution,
which becomes particularly useful when the system of interest is sensitiveto
exchange broadening in proton resonances.
38
Lastly, when dealing with high-Q
detection, such as in cryogenic probes,
13
C-detected experiments are less
sensitive to the salt concentration of the sample solution than
1
H-detected
experiments.
39
The results shown demonstrate that, the inherently lower
sensitivity of
13
C-detected experiments relative to
1
H-detection is partially
compensated by the ability to work at high salt concentrations. This therefore
significantly extends the range of sample conditions under which
13
C-detection
becomes competitive.
