The STD method was reported as a fast method to screen compound libraries for binding to proteins, with dissociation constants KD between 10 nM and µM. STD experiments rely on magnetization exchange from the protein-bound state to the free state of the ligandThe method consists in applying radio-frequency irradiation to selectively saturate the protein NMR signals. The saturation then spreads within the protein via the NOE and spin diffusion. In the presence of a ligand and under fast exchange conditions, the saturation will also spread to the ligand through intermolecular NOEs. In practice, spectra are recorded with and without saturation of the protein protons. In the case of saturation “on resonance”, the signals of the compounds that bind to the protein will be saturated (and thus attenuated). By subtracting the spectrum corresponding to the receptor saturation “off resonance”, one observes only the signals of the binders, whereas the signals of the protein and non-binders are subtracted.
STD experiments are performed in a large excess of the ligand. The protein-to-ligand ratio typically ranges from 1:100 to 1:1,000 for large proteins (larger than 80 kDa). The protein concentration depends on the molecular weight of the macromolecule (the larger the protein, the lower its concentration) but usually ranges from 0.1 mM to 100 pM. Typical saturation times are 1–2 s.