Chemistry Reference
In-Depth Information
Chemical shift mapping provides information about the location of the metal
binding sites and has been used to identify the metal coordination for numerous
metalloproteins [
15
,
106
-
108
]. For example, addition of Cu
+
to human Cox17 induced
significant chemical shift variations over residues Lys20 and Ala24, and the appearance
of the NH signals of Cys22-Cys23, which is thought to serve as the Cu
+
binding motif
[
107
]. However, this approach usually has to be used in combination with other
physical techniques or biological approaches, e.g., mutagenesis to specify the metal
binding sites, since the chemical shift perturbations mainly stem from either direct
binding or conformational changes caused by the metal ions. Binding of Ni
2+
to
H.
pylori
HypA led to disappearance of signals of Glu3 and Asp40 in the 2D
1
H-
15
N
HSQC spectrum. When combining with mutagenesis study, side-chain 2D
1
H-
15
N
HMQC, UV absorption, as well as CD, it was proposed that Ni
2+
coordinates with His2
(side-chain N
d
), His2 (backbone N), and the backbone nitrogens of Glu3, and Asp40
with a square-planar geometry [
15
]. Such a binding also induced structural changes
which were thought to be important for its downstream receptor's recognition [
15
].
Histidine often serves as a metal binding ligand in metalloproteins and can
provide both backbone and side nitrogens to coordinate with metal ions such as
Zn
2+
and Ni
2+
. It has been shown that different tautomeric forms of histidine
imadazole rings have different, distinguishable signal patterns in a long-range 2D
1
H-
15
N HMQC spectrum [
109
], metal coordination often causes recognizable
changes in the NMR spectrum of histidine side-chains, and imadazole nitrogen
atoms involved in direct metal coordination have specific chemical shift [
15
,
40
,
98
,
110
]. This technique has been extensively used to identify Zn
2+
binding. The
chemical shifts observed for the unprotonated imadazole nitrogen atoms of a zinc
finger domain Hdm2(429-491) appeared at ca. 215 ppm. The cross-peak pattern in
1
H-
15
N HMQC spectrum (with
2
J
HN
) of zinc bound Hdm2 (429-491) showed that
His452 and His457 assumed different tautomeric forms with the former being N
e
2
-
protonated and the latter N
d
1
-protonated with chemical shifts around 170 ppm,
which demonstrated that Zn
2+
coordinated to both His452 and His457 via the N
e
2
and N
d
1
respectively [
98
]. More interestingly, a comparison of the 2D
1
H-
15
N
HMQC spectra of Zn
2+
-bound proteins with
113
Cd
2+
-bound proteins, as shown in
Fig.
6
, allows one to observe the
15
N-
113
Cd coupling, which assists identification of
overlapping of histidine side-chains that bind to metal ions [
40
]. The HMQC
spectrum of the zinc-bound domain, shown as black in Fig.
6a
, clearly shows two
of the
15
N resonances His42 and His40 shifted downfield as a result of zinc
coordination. Based on the pattern of the cross-peaks, His42 is in the
tautomeric
state indicative of zinc coordination to the N
d
1
of His42; Fig.
6b
. Such a method
cannot be used to assign the nitrogen atom of His40 due to overlapping of the H
d
2
and H
e
1
resonances of His40. The HMQC spectrum of
113
Cd bound protein, shown
as red in Fig.
6a
, clearly shows coupling between
113
Cd and the N
d
1
of His42
observed on H
e
1
, suggesting a covalent bond between them. Importantly, the
pattern of the connectivities and the coupling observed to both H
d
2
and H
e
1
from
the
113
Cd allows the unambiguous assignment of the
e
tautomer for His40, with the
metal coordination via the N
e
2
of the side-chain; Fig.
6b
[
40
]. The side-chain 2D
1
H-
15
N HMQC spectrum of histidine has also been used to identify Ni
2+
binding
d
