Chemistry Reference
In-Depth Information
genetically encoded Co
2+
-binding amino acid [
140
]. Moreover, PCSs can also be
used as restraints in molecular dynamics [
141
,
142
].
13
C-Detected Protonless NMR
4.2
Direct detection of heteronuclei, in particular
13
C, offers a valuable alternative to
1
H detection in the study of biological macromolecules [
143
-
145
] as well as
paramagnetic proteins [
136
,
145
-
147
]. The recent development of high magnetic
fields as well as the availability of cryogenically cooled probe heads has improved
13
C sensitivity significantly, which has greatly stimulated research using
13
C-detected experiments on enriched samples to study biological macromolecules
[
148
]. The
13
C detection takes advantage of the slower relaxing
13
C spins and
overcomes the drawbacks produced by the fast
1
H transverse relaxation, which
leads
1
H signals to broaden beyond detection limits for large proteins. Such an
approach is particularly useful in paramagnetic systems since
13
C direct detection
is less affected by the paramagnetic center than
1
H owing to the lower
13
C
gyromagnetic ratio, which decreases the paramagnetic dipolar contributions to its
relaxation by a factor of around 16 (
g
H
)
2
[
136
,
146
,
149
].
In
13
C direct detection experiments, several approaches were used to achieve
“virtual” decoupling (to remove homonuclear one-bond carbon-carbon couplings)
such as IPAP schemes (in-phase anti-phase) [
150
-
152
], in which two FIDs for each
increment are recorded and stored separately, one for in-phase and another for anti-
phase, the two components being combined to remove the splitting. An alternative
is S
3
E schemes [
152
,
153
] (spin-state selective excitation), in which two different
experiments are performed with one being absorptive and another dispersive. One
or more of these building blocks (IPAP and S
3
E) can be implemented in any
experiments based on
13
C direct detection. A set of
13
C based experiments, which
can be used for the assignment of backbone and side-chains of
13
C/
15
N labeled
proteins, is now available and summarized in a recent review [
144
]. The sequence-
specific assignment was achieved by CACO and CANCO, which provide the
correlation of each CO to the two neighboring C
a
nuclei; CACO, CBCACO,
and
13
C-
13
C TOCSY can provide spin-system assignment [
143
,
154
,
155
]. The
CON-IPAP experiment is used to correlate backbone nitrogen with CO through
the one-bond C
0
-N coupling [
149
]. The
13
C-
13
C NOESY experiments based
on dipole-dipole interaction with longitudinal magnetization transfer represent a
valuable alternative to COSY experiments based on scalar couplings to detect C-C
one-bond correlation for large macromolecules and paramagnetic metalloproteins
[
149
,
156
,
157
]. The
13
C-
13
C NOESY with direct
13
C detection is exploited as
a valuable tool to extend the assignment to side-chains in large molecules such as
C
0
-C
b
if mixing times is long enough (e.g., 800 ms) [
156
]. The correlations between
nuclei not directly bound and not mediated by spin diffusion in
13
C-
13
C NOESY
spectra are identified, which would represent a breakthrough of structure determi-
nation of large macromolecules in solution by providing distance constraints.
g
C
/
