Chemistry Reference
In-Depth Information
to pseudo-contact shifts, and Curie-spin relaxation is negligible [
37
]. The advantage
of their employment for pure PRE measurements is the fast and straightforward
nature of the method: the resonance assignments in the correlation spectra, known
from through-bond scalar triple resonance experiments, are not perturbed by para-
magnetic modification and the high sensitivity results in an experimental time of
5-40 min on a typical protein sample (the same advantages we discussed for CSP
method, as both are based on the same types of data acquisition - HSQC
experiments). The potential problems are associated with intrinsic flexibility/rotation
of either the paramagnetic tag itself or its attachment to the protein, resulting in the
time average distances sampled over all possible conformations. Thus certain caution
is required for incorporation of the derived distances as the restraints for structure
calculations, where a paramagnetic center, for example, can be treated as an ensemble
average rather than a fixed point [
38
]. The other possibility for highly flexible systems
is to use PRE data loosely, as a guide, rather than major geometric restraints, for
example, in structure determination of the complex when the orientation of the
peptide, which could be labeled by a paramagnetic tag, in a particular binding site
needs to be addressed [
39
,
40
]. The potential ability to study transient low population
intermediates in macromolecular interactions is conceivably one of the most exciting
PRE implementations in structural biology. These illusive species are rarely accessi-
ble by other than NMR biophysical techniques. In an exchanging system the observed
PRE measured on the resonance of the major species can be modulated by the minor
species to the extent depending upon the rate of exchange [
41
], with the fast exchange
allowing one to characterize structurally populations comprising as low as 1%. The
example illustrating PRE potential to demonstrate the existence and visualize the
distribution of an ensemble of transient non-specific intermediates in addition to
specific complex formation has been presented by Clore and colleagues for a
bacterial phosphotransferase system [
42
]. Thus, it has been proven that PRE data is
highly sensitive asserting weak interactions characterized by large k
off
rate and is
salutary for structural analysis of weak PPIs.
1.4.2 The PCS Effect
PCS is a phenomenon that is only observable for paramagnetic systems with aniso-
tropic unpaired electrons such as those found in Dy
3+
,Tb
3+
,andFe
3+
lanthanide ions,
characterized by an anisotropic electron
g
-tensor. In general, if the
g-
tensor is aniso-
tropic, than the magnetic susceptibility tensor (usually referred to as the
-tensor) is
anisotropic as well. The magnitude of the PCS depends on the orientations of the
vectors connecting the lanthanide ion and affected nuclei with respect to direction of
the external magnetic field. These orientations are not averaged because tumbling in
aqueous solution appears to be non-isotropic due to the effect of large
w
-tensor
intrinsic for these paramagnetic species. This large magnetic susceptibility tensor
provides enough energy to overcome random Brownian motion and to generate
w
