Chemistry Reference
In-Depth Information
associated ligand (Met or others) which is slightly different for these proteins.
Nevertheless, such a subtle difference can be faithfully reflected by the chemical
shifts of
199
Hg. The chemical shift map of
199
Hg can be derived based on various
model complexes and proteins; Fig.
5b
. The large chemical shift dispersion for
199
Hg allows clear differentiation between a variety of M(SR)
n
environments.
Given that Hg
2+
is readily exchanged for the native metal ion in many copper,
zinc, and iron metalloproteins [
96
],
199
Hg NMR methods can play an important role
in structural, spectroscopic, and chemical studies of metalloproteins and metal
binding domains where the tertiary structure of the folded proteins dictates the
geometry of the metal ion.
3.2 Chemical Shift Perturbation
Protein NMR chemical shift is highly sensitive to the exact environment of the atom
and can provide valuable insights into structural features including metal ligation.
For examples, chemical shifts of
13
C
b
for the zinc bound cysteines (ca. 34 ppm) are
significantly downfield shifted relative to those of non-metal bound cysteines
(ca. 27 ppm) [
97
]. Such an index has often been used to discriminate zinc bound
cysteine residues [
15
,
98
]. The chemical shift perturbations upon metal ion binding
can be used to estimate the affinity, stoichiometry, and kinetics of metal binding,
and, moreover, they can be used to identify metal coordination environments. This
approach is usually denoted as chemical shift mapping, which has been widely used
to study protein-protein interaction [
99
,
100
].
1
H NMR and
1
H-
1
H TOCSY have
been used to identify the types of residues binding to metal ions for unlabeled small
metalloproteins [
101
-
103
]. However, two dimensional HSQC, especially
1
H-
15
N
HSQC, is often employed in studies of metal coordination environments in
metalloproteins due to the fact that it is well resolved in comparison with 2D
1
H-
13
C HSQC. The identity of each cross-peak in the 2D
1
H-
15
N HSQC spectra
is assigned based on a series of triple resonance experiments. Binding of metal ions
would lead to the appearance of new peaks or disappearance of original peaks
depending on the exchange rates of the apo- and metal-bound forms on the NMR
time scales [
99
,
104
]. The chemical shifts perturbation (CSP) can be followed in
titration experiments, where the concentration of diamagnetic metal ion is increased
gradually. When heteronuclear data are available, the binding site is usually
predicated by the combined chemical shift perturbation
Dd
comb
, which has been
shown to be a more reliable approach to evaluate titration data quantitatively [
105
].
Although several approaches are available to obtain this value as summarized
previously [
105
], in practice, the weighting for
1
H and
15
N is considered to be the
same, and the
Dd
comb
is usually quantified by the following equation:
s
Dd
2
HN
þ
2
N
1
25
Dd
Dd
comb
¼
:
2
