3.5.2 STD
Signal in the STD spectrum arises from the transfer of saturation
from the protein directly to bound ligands; this saturation then
persists after dissociation and causes the build-up of a population of
ligand with saturated resonances, which is manifested as a positive
signal in the saturation transfer difference experiment [25]. The
signal in the STD spectrum therefore reflects the bound population
of the ligand and indicates which molecules are binding to the
protein. After displacement by a potent competitor, the STD signal
for a ligand that is binding specifically to the protein will be substantially reduced.
However, artifactual signal can occur in the STD following
direct saturation of ligand resonances, particularly where the ligand
contains aliphatic groups with chemical shifts that are relatively
close to the saturation frequency (within 1 ppm or so, depending
on the shape used for selective saturation of the protein). In this
case, a large STD signal is typically seen for the ligand (particularly
for the aliphatic resonances that are directly saturated), and this
STD signal is not perturbed by the addition of a potent competitor
molecule.
3.5.3 Water-LOGSY
In the water-LOGSY experiment, magnetization is transferred from
excited water molecules directly to the compound [26]. If the
compound is free in solution, these water molecules form the
hydration shell, and the rapid tumbling of the compound and
water molecules gives rise to a negative signal. If the compound is
bound to a protein, the primary water molecules giving rise to
signal are those present in and around the ligand-binding site on
the protein. Since this system is tumbling slowly, a signal with a
positive sign is observed; this positive signal persists into solution
after dissociation from the protein.
The resultant signal is therefore a function of the free unbound
population (with negative signal that depends on the nature of the
hydration shell of the free molecule) and the population that has
been bound to the protein (with positive signal that depends on the
presence of waters associated with the ligand in the bound state).
This means that compounds that bind may still give rise to a
negative signal if the contribution from the free state is large
and/or that from the bound state is small.
In our experience, the most reliable interpretation of waterLOGSY spectra relies on perturbation of the free and bound populations following the addition of a potent competitor. Displacement
of the fragment from the binding site by the competitor results in
an increase in the unbound population, which gives rise to a more
negative signal regardless of the magnitude of the contributions
from the free and bound states of the ligand. This competition step
is not strictly required for interpretation of the water-LOGSY
spectra, but does significantly increase the confidence level of the
result.
262
Ben J. Davis
Signal in the STD spectrum arises from the transfer of saturation
from the protein directly to bound ligands; this saturation then
persists after dissociation and causes the build-up of a population of
ligand with saturated resonances, which is manifested as a positive
signal in the saturation transfer difference experiment [25]. The
signal in the STD spectrum therefore reflects the bound population
of the ligand and indicates which molecules are binding to the
protein. After displacement by a potent competitor, the STD signal
for a ligand that is binding specifically to the protein will be substantially reduced.
However, artifactual signal can occur in the STD following
direct saturation of ligand resonances, particularly where the ligand
contains aliphatic groups with chemical shifts that are relatively
close to the saturation frequency (within 1 ppm or so, depending
on the shape used for selective saturation of the protein). In this
case, a large STD signal is typically seen for the ligand (particularly
for the aliphatic resonances that are directly saturated), and this
STD signal is not perturbed by the addition of a potent competitor
molecule.
3.5.3 Water-LOGSY
In the water-LOGSY experiment, magnetization is transferred from
excited water molecules directly to the compound [26]. If the
compound is free in solution, these water molecules form the
hydration shell, and the rapid tumbling of the compound and
water molecules gives rise to a negative signal. If the compound is
bound to a protein, the primary water molecules giving rise to
signal are those present in and around the ligand-binding site on
the protein. Since this system is tumbling slowly, a signal with a
positive sign is observed; this positive signal persists into solution
after dissociation from the protein.
The resultant signal is therefore a function of the free unbound
population (with negative signal that depends on the nature of the
hydration shell of the free molecule) and the population that has
been bound to the protein (with positive signal that depends on the
presence of waters associated with the ligand in the bound state).
This means that compounds that bind may still give rise to a
negative signal if the contribution from the free state is large
and/or that from the bound state is small.
In our experience, the most reliable interpretation of waterLOGSY spectra relies on perturbation of the free and bound populations following the addition of a potent competitor. Displacement
of the fragment from the binding site by the competitor results in
an increase in the unbound population, which gives rise to a more
negative signal regardless of the magnitude of the contributions
from the free and bound states of the ligand. This competition step
is not strictly required for interpretation of the water-LOGSY
spectra, but does significantly increase the confidence level of the
result.
262
Ben J. Davis