Chemistry Reference
In-Depth Information
However, the sensitivity for
13
C direct detection is required to be improved before
long-range correlations to obtain
13
C-
13
C distance constraints can be used in
structural characterization of large macromolecules.
13
C direct detection has been successfully applied to paramagnetic proteins,
where the contribution to line broadening coming from the paramagnetic center
is so large that
1
H signals around the metal ion are beyond detectable limits
[
136
,
144
,
147
,
149
,
158
]. Such a technique can also be used in generation of
paramagnetism-based restraints including PCS, PRE, and RDC [
148
,
159
-
161
]. It
has been demonstrated that
13
C directly detected spectra provide an alternative
method for the measurement of RDC with precision as good as that from
1
H
detection, but with additional advantage for measuring those broad resonances in
1
H detection [
148
]. Direct detection of
13
C intrinsically offers a way to detect
resonances close to the metal ion where
1
H resonances are too broad to be detected.
Indeed, with the aid of a
13
C direct detection approach,
13
C resonances as close
as 6
˚
from the metal ion are detected for CopC, a Cu
2+
binding protein involved in
copper homeostasis, whereas no
1
H resonance can be detected within a sphere of
11
˚
from the metal due to fast relaxation caused by paramagnetic Cu
2+
[
136
].
Incorporation of heteronuclear paramagnetism-based restraints, e.g., PCSs and
longitudinal relaxation rate enhancement, allows CopC structures to be resolved
with the RMSD of Cu
2+
determined only by the paramagnetism-based constraints
of 1.1
˚
[
136
]. The
13
C direct detection technique has also been used for residue-
specific assignments of resonances, in particular those near paramagnetic centers
(e.g., Ni
2+
and Fe
3+
) such as in a 20-kDa Ni-containing enzyme, acireductone
dioxyhenase (ARD) [
162
], and oxidized human [2Fe-2S] ferredoxin [
146
], as
well as a 19-kDa Fe
3+
hemophore HasA [
147
]. In many paramagnetic systems,
the longitudinal relaxation rates are influenced to a smaller extent than the trans-
verse relaxation rates. The
13
C-
13
C NOESY experiments are therefore a useful
approach to overcome the quench of scalar coupling based transfer, in particular for
large macromolecules. The use of
13
C direct detected experiments, e.g.,
13
C-
13
C
COSY,
13
C-
13
C NOESY, and
13
C-
13
C COCAMQ, allows
13
C signals as close as
4
˚
to Cu
2+
to be detected in oxidized monomeric copper/zinc superoxide
dismutase (SOD) [
149
]. The advantage of
13
C-
13
C NOESY experiments for higher
molecular weights was seen by comparison of the protein SOD [
149
,
156
]. All
of the expected C
-CO connectivities were detected with higher intensity in the
dimeric protein than in the monomeric state. In addition, most of the two bond
CO-C
a
cross-peaks were observable for the dimeric SOD when the long mixing
times were used [
156
]. Interestingly, the intrinsic asymmetry of a
13
C-
13
C COSY
experiment allows the coordinating residues of paramagnetic metal ions to be
identified easily, providing a unique method to distinguish between monodentate
and bidentate coordinating side-chain carbonyls [
163
]. Significantly, the
13
C-based
strategy in combination with solid-state NMR led to partial sequence-specific
(35%) and side-chain assignments for the iron storage protein, ferritin, a very
large protein with a molecular mass of 480 kDa and 24 subunits [
164
,
165
]. The
solution
13
C-
13
C NOESY spectra for side-chain observation has provided the
identification of an iron channel that guides the direction transport of the multimeric
b
