Biomedical Engineering Reference
In-Depth Information
deuterium-to-proton exchange process is often unsuccessful leading to
incomplete assignment of the interior of the molecules. Thus, the experiment
can be a valuable complement to conventional proton-detected triple-
resonance experiments. Recently, an interesting way to jointly use these two
types of experiments was proposed by utilising multiple receivers.
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In this
approach, the weak signal that remains after
13
C-detected experiments
(referred to the '
13
C-afterglow') can still be measured with high sensitivity by
proton detection.
Assuming no relaxation, the signal intensities in experiments using low-c
nuclei are expected to be lower. However, as we directly compared
15
N-direct
detection against
13
C-direct detection, the sensitivity losses due to the lower c
(in this case
15
N) nuclei can be compensated by the slower transverse
relaxation and an appropriate experimental design. For an appropriate
experimental design, both labelling strategies and pulse sequences need to be
considered based on their relaxation properties (composition of DD and CSA
relaxation etc.) as well as the spin-spin coupling patterns. This is especially
true for
13
C
a
, which suffers from a complicated homonuclear spin-spin
coupling pattern when conventional uniform
13
C-labelling is used. The
13
C-
12
C alternate labelling with deuteration using site-specific
13
C-labelled
amino acid precursors, such as glycerol and pyruvate has been proposed for
13
C
a
-detection to avoid this problem.
50
This labelling strategy also changes the
spin-spin coupling networks within a protein, and makes small couplings
available for transfers that are not usable in uniformly
13
C-labelled samples, as
shown for CACA-TOCSY. As the main drawback of the
13
C-
12
C alternate
labelling procedure is that not all residues are fully
13
C
a
-labelled, therefore,
complementary use of the IPAP-type decoupling combined with uniform
13
C-
labelling and further development of labelling strategies might be beneficial.
The low sensitivity of low-c nuclei can be improved by non-linear sampling.
Furthermore, paramagnetic relaxation reagents allow shorter repetition delays,
which can increase the sensitivity per unit time significantly.
To determine a 3D structure solely from low-c nuclei detection is more
challenging since experiments that rely on DD cross-relaxation may not work
efficiently. Experiments utilising RDCs as well as PRE have been proposed to
obtain structural information. Along these lines, measuring pseudo-contact
shifts (PCSs) would be quite interesting and could provide long-range
information relative to paramagnetic centres or lanthanide ions bound to
engineered binding sites, which obviously leaves room for exploration.
Acknowledgements
Research described here was supported by the National Institute of Health
(grants AI037581, GM047467 and EB002026), the Ministry of Economy,
Trade and Industry (METI) of Japan, and the Japan New Energy and
Industrial Technology Development Organization (NEDO).