Chemistry Reference
In-Depth Information
where
I
dia
and
I
para
are the peak intensities for the diamagnetic and paramagnetic
states, respectively. The choice of time points is important to minimize the error.
For example, if the range of
G
2
rates is 0-75 s
1
, a second time point T
b
should be at
ca. 1.15/(
R
2
,
dia
+ 50) s, representing a reasonable choice [
115
,
116
].
For isotropic metal ions such as Mn
2+
and Gd
3+
, the Curie-spin relaxation that could
potentially exhibit significant cross-correlation with other relaxation mechanisms is
negligible for medium-size macromolecules [
116
], and the Solomon relaxation is
predominant. PRE analysis in such a system is thus simple. The PRE has been used
extensively in metalloproteins that possess a rigid intrinsic paramagnetic center
[
117
-
119
]. Such a strategy has also been extended not only in the NMR structure
determination of non metalloproteins [
120
-
122
], in which paramagnetic metal ions
(Mn
2+
or Gd
3+
) or nitroxide radicals were conjugated through appropriate chemical
modification [
123
], but also in the characterization of protein-protein/nucleic acid
complexes [
124
-
126
] and membrane-proteins [
127
], in particular in transient macro-
molecular interactions [
115
,
128
-
131
].
PCSs are precious sources of structure information and are observed only in
paramagnetic systems with anisotropic unpaired electrons, e.g., Dy
3+
,Tb
3+
,and
Fe
3+
. The magnitude of the PCS,
pcs
, is calculated using the following equation [
132
]:
d
1
12
3
2
Dw
rh
sin
2
pcs
r
3
3cos
2
d
¼
Dw
ax
ð
y
1
Þþ
y
cos 2
'
p
1
2
ðw
xx
þ w
yy
Þ
Dw
ax
¼ Dw
zz
and
Dw
rh
¼ w
xx
w
yy
where
r
is the distance between the metal ion and the nuclear spin,
are the
angles describing the position of the nuclear spin with respect to the principle axes
of the magnetic susceptibility tensor
y
and
'
Dw
rh
are the axial and rhombic
components, respectively, of the magnetic susceptibility tensor.
The PCS are manifested by large changes in chemical shifts of the nuclear spins
that are exposed to the paramagnetic metal ions and arise from through-space
dipolar interactions with rapidly relaxing unpaired electrons. The PCS displays an
r
3
distance dependence, in contrast to the
r
6
dependence for the PRE, which
results in a relatively long distance range for the PCS to be detected (ca. 40
˚
w
, and
Dw
ax
and
for
Dy
3+
)[
133
]. In general, the
pcs
values can be measured after the complete
assignment is obtained for the
1
H-
15
N HSQC spectra of the both the diamagnetic
and the paramagnetic samples, and are calculated as the difference between the
chemical-shift values observed for the nuclei in a paramagnetic system and in a
diamagnetic analog. The
d
pcs
-derived restraints alone cannot be used to solve the
structures. Instead, the PCSs have to be incorporated with NOEs and dihedral-angle
restraints to determine structures of proteins or to refine protein structures. The
first example of using PCS in a structure refinement was reported on a low-spin
Fe
3+
heme protein [
134
]. Such a strategy has been extended not only in studies of
paramagnetic proteins [
135
,
136
], but also in non-metal binding proteins [
137
,
138
],
which were labeled by paramagnetic metal ions such as lanthanides [
114
,
139
]or
d
