Chemistry Reference
In-Depth Information
preferable orientation or alignment of the macromolecule containing the paramagnetic
tag. This phenomenon causes changes in chemical shift of the affected nuclei which
are sufficiently close to the paramagnetic center (reviewed in [
43
]). Importantly, the
PCS displays basic
r
3
distance proportionality in contrast to the
r
6
dependence
for the PRE or NOE. In theory, this will define a relatively longer experimentally
attainable distance range, extending it up to, for example, ~40
˚
for Dy
3+
in
w
metalloproteins. In practice, the principal axis of the
-tensor is not rigidly fixed
within the molecular frame when an extrinsic metal ion is attached to a macromole-
cule using a chelator with a flexible linker, causing significant reduction in the
magnitude of the PCS because of
w
-tensor principal axes fluctuations within the frame
of the macromolecule. From the perspective of studying wPPIs, PCS restraints can be
generated using a
15
N-labeled and/or
13
C-labeled protein bound to an unlabeled but
paramagnetically tagged partner.
15
Nand/or
13
C-HSQC experiments then need to be
recorded for both the paramagnetic and diamagnetic states of a sample and chemical
shift changes should be extracted from the spectra [
31
]. However, to use PCS data,
one first has to define the tensor describing the anisotropic magnetic moment of the
paramagnetic center [
44
]. When the structures of the individual proteins are known,
PCS data can be combined with rigid-body docking to produce a model for a protein
complex. This approach has been proven successful in determination of a 30-kDa
complex between the
subunits of
Escherichia coli
polymerase III [
45
], where
the active-site bound Mg
2+
/Mn
2+
pairs were exchanged with paramagnetic Dy
3+
or
Er
3+
and corresponding
15
N-HSQC spectra of the diamagnetic apo-complex and
paramagnetic-ion-bound complexes were compared. An analogous approach taking
advantage of the intrinsic iron-binding capability of cytochrome f has been used
earlier to define the structure of its transient complex with plastocyanin: conveniently,
iron in its oxidized Fe
3+
y
and
e
form is paramagnetic while in the Fe
2+
form it displays
a diamagnetic nature [
46
].
2 Conclusions
While tight protein interactions can be addressed experimentally by many
techniques, including X-ray crystallography, the vast majority of these approaches
fail or become unreliable when the interactions are weak. Solution NMR spectro-
scopy is unique among the structural techniques, permitting the characterizing of
weak interactions and providing structures of weak protein-target complexes. If
such interactions involve small molecules, NMR can be employed for optimization
and development of drug-leads. In the current post-genomic era, the NMR methods
we have highlighted in combination with functional and computational approaches
hold significant promise for characterizing the plethora of weak protein complexes
that regulate cellular events, thereby providing an unbiased and comprehensive
view of how proteins function in living cells.
