Chemistry Reference
In-Depth Information
transfer can occur between the target and ligand spins in transient forming
complexes through the NOE.
In STD experiments, a selective radio frequency pulse is applied in a spectral
region where no ligand resonances are expected, but the protein is completely
saturated due to its larger line width and spin diffusion. Moreover, a ligand that
binds to the protein will also be saturated. The degree of ligand saturation
naturally relies on the residence time of the ligand in the receptor binding site.
Thus, after dissociation, the ligand can be observed in the free state by a reduction
of signal intensity compared to a reference spectrum with irradiation far away
from any resonance. Therefore, subtracting a spectrum, in which the receptor is
saturated, from one without protein saturation produces a difference spectrum in
which only the signals of the ligand(s) remain [3, 27, 32].
On the other hand, STD experiments are strongly K
D
dependent. In other words, if
binding is very tight, the saturation transfer to the ligand molecules is not very
efficient. This is common for K
D
values lower than 1 nm. On the other hand, if the
K
D
values are 100 nM or higher, fast exchange of free and bound ligands leads to
a very efficient buildup of saturation of the ligand molecules in solution. It is clear
that the observed intensity of the signals arising from the ligand in the STD NMR
spectrum is not proportional to the binding strength. STD NMR effects depend
largely on the off rate, since larger off rates should result in larger STD signals.
However, when binding becomes very weak, the probability of the ligand being in
the receptor site becomes very low. This results in weak STD signals. STD NMR
spectroscopy can be used from very tight binding up to a K
D
of about 10 mM [3].
Moreover, the intensity of the STD signals is also dependent on several
parameters that can be optimized, such as the receptor concentration, the ligand-
receptor concentration ratio and the saturation time [1]. In some cases, the need of
a large ligand excess allows STD measurements with very low protein
concentrations. A discussion about theses parameters, as well as STD
amplification factor, has been reported by Mayer & Meyer [33-36].
One of the advantages of the STD method is that it can be incorporated into two-
dimensional experiments, such as two-dimensional STD-TOCSY or STD-HSQC
