Peptide-Based Drug Design

NMR of Peptides 101
4.4.2. Saturation Transfer Difference (STD) NMR
The STD NMR experiment employs a train of low-power pulses to selectively
saturate proton resonances of a receptor (63). If the receptor exceeds
∼10 kDa, it is usually subject to spin diffusion, which causes the saturation to
spread uniformly over all resonances of the receptor. The saturation can then be
transferred to the parts of a small molecule directly bound to the receptor, as
illustrated in Fig. 9. The saturation of the bound molecule’s proton resonances
near the binding site is detected after dissociation of the ligand into solution as
attenuation of the respective signals.
A typical STD NMR spectrum is achieved by subtracting the so-called onresonance
spectrum from the off-resonance spectrum, where no saturation is
applied to the protein. The latter spectrum is usually acquired by applying
saturation pulses far outside the spectral window of the receptor to maintain
relaxation and thermal equality for the experiments. To average out effects
occurring over time, the two spectra are usually recorded in an interleaved
fashion. The generation of the difference spectrum is highlighted in Fig. 10.
STD NMR experiments can be used to determine both the orientation of a ligand
in a binding site and the dissociation constant of a ligand-receptor complex via
titration experiments. STD NMR can be performed as a 1D experiment or as 2D
correlation spectra if more dispersion is required (63,64).
Fig. 9. Schematic representation of the saturation transfer from receptor to a bound
ligand. The protons of the receptor are saturated by a train of selective pulses. Upon
binding (middle) the saturation is transferred to protons of the ligand. The different
emphasis on the ligand protons represents the effectiveness of the transfer depending
on proximity of ligand protons to protons on the receptor surface. This information is
transferred back into solution upon dissociation of the complex.