Biomedical Engineering Reference
In-Depth Information
was sufficient to establish complete sequence-specific backbone and side-chain
assignments of small-to-medium-size proteins.
The success of all these deconvolution techniques in decoupling of
1
J
C9Ca
is
at least partially due to the fact that the coupling is very uniform and C9 and
C
a
chemical shifts never overlap. On the other hand, direct C
˜
detection is
complicated due to the large scalar couplings which have different values, y55
Hz for C9 and y35 Hz for C
b
. This causes splitting of C
a
resonances into
quartets. In addition, C
a
and C
b
chemical shifts partially overlap and designing
a pulse perfectly selective to one of these nuclei is impossible. However,
13
C
a
nuclei have a small CSA and relax slower if samples are deuterated. Thus,
deuterated
13
C
a
might be the better nuclei for
13
C-detection experiments in
large and/or fast-relaxing systems. To obtain a single high-resolution line in
13
C
a
-detected experiments, most of the techniques discussed above for
13
C9
could work in principle, however, more complicated schemes are generally
needed and the sensitivity gain may not be optimal.
As for the IPAP-type decoupling, two IPAP blocks, one for
1
J
CaC9
, the other
for
1
J
CaCb
coupling are combined with each other in a concatenated fashion. In
this double IPAP (DIPAP) scheme, a total of four FIDs for each increment,
which contains IP-IP, AP-IP, IP-AP, and AP-AP information (the former is
for
1
J
CaC9
and the latter is for
1
J
CaCb
), should be recorded. These are combined
and shifted to restore a single line in the middle between split lines. The
duration of the DIPAP block is rather long, 14 ms, reflecting weaker
1
J
CaCb
coupling. In addition, resonances from Gly, Ser and some high-field C
a
nuclei
can be significantly reduced by the
13
C
a
selective pulses needed in the DIPAP
scheme.
Recently, we have introduced the use of
13
C-
12
C alternate labelling to
overcome one-bond
13
C-
13
C coupling in C
a
-detected triple-resonance experi-
ments.
50
This strategy uses an isotope-labelling scheme similar to the
procedure established by LeMaster.
52
This procedure was recently also used
to observe long-range
13
C-
13
C distance correlations of up to 7
˚
in solids.
68
The strategy enables alternate
13
C-
12
C-labelling at most positions by
expressing the protein in E. coli using either [2-
13
C] glycerol or [1,3-
13
C]
glycerol as carbon source [Figure 2.2(A) and (B)]. One can also use a site-
specific
13
C-labelled pyruvate or acetate instead of glycerol to obtain a similar
labelling pattern.
Figure 2.2(C) shows the H
a
-C
a
region in a
1
H-
13
C HSQC spectrum of a
uniformly
13
C-labelled SH3 domain and illustrates the spectral complexity in
the indirect dimension due to
13
C
a
-
13
C9 and
13
C
a
-
13
C
b
couplings. The same
protein labelled with [2-
13
C] glycerol has well-resolved singlets indicating that
neighbouring carbons are not
13
C-labelled (Figure 2.2(C) right). Only the
valines and isoleucines (red arrows) exhibit doublets due to the
13
C
a
-
13
C
b
couplings, as is expected from the metabolic pathways.
Figure 2.2(D) classifies the
13
C/
12
C labelling ratios at the C
a
positions in the
13
C-
12
C alternate labelling scheme. [2-
13
C] and [1,3-
13
C] glycerol labelling
yielded inverse labelling patterns. As expected from amino acid synthetic
