Chemistry Reference
In-Depth Information
[3, 36, 37]. In STD experiments, the degree of saturation of ligand resonances
depends on the distance of the protons involved, which are saturated to a higher
degree when in close proximity to the receptor molecule, thus leading to a
stronger STD signal. This information can be used to perform a mapping of the
binding epitope (group epitope mapping) [35]. This is important for the directed
developments of drugs [4]. Actually, there are several papers in the literature
describing STD-NMR as a tool in order to identify the binding epitope, obtain the
binding constants, characterize ligand binding to viruses, membrane-integrated
proteins and immobilized proteins [3].
WaterLOGSY
The WaterLOGSY (Water-Ligand Observed
via
Gradient SpectroscopY)
experiment is a variation of magnetization transfer between protein and ligand,
employing the large bulk water magnetization to transfer magnetization
via
the
protein-ligand complex to the free ligand, in a selective manner. Magnetization is
transferred from water to the protein and to the ligand. It can also be transferred
directly from bulk water to the ligand without the pathways through the protein.
Meantime, signals of non-binding ligands have opposite sign compared to signals
of ligands that bind with high affinity, allowing fast distinction between bound
and not bound molecules to the protein [15, 26].
The method is more efficient for ligands displaying dissociation constants in the
µM to mM range. Normally, an excess of ligand (10 to 20 fold) with respect to the
protein concentration must be used. In addition, it can also be incorporated into
two-dimensional NMR experiments. In some cases, optimization of parameters
can be obtained by raising the temperature, adding small amounts of organic
solvent or by changing the ionic strength [4].
WaterLOGSY experiments have a great potential in primary NMR screening,
since the experiment is very sensitive and requires only very low protein and
modest ligand concentrations. One disadvantage of this method is that binding and
non-binding compounds give signals of opposite phase, resulting in complications
concerning crowded spectra, which is very common to combinatorial libraries [11,
26].
