Abstract
We have studied the potential of saturation transfer nuclear magnetic resonance (NMR) experiments in different mode to screen compound mixtures for binding activity and to characterize binding epitopes on the ligand. We have developed a protocol based on the transfer of saturation from the protein to bound ligands which by dissociation is moved into solution where it is detected (Fig. 1; Mayer and Meyer 1999; Peters and Meyer). By subtracting a spectrum, where the protein is saturated from one without protein saturation, a spectrum is produced where only signals of the ligand(s) remain in the difference spectrum. The irradiation frequency is set to a value where only protein resonances and no resonances of free ligands are located. Usually, irradiation frequencies around 1.5 ppm are practical because no ligand resonances are found in this spectral region, whereas the significant line width of protein signals still allows selective saturation. If the ligands show no resonances in the aromatic spectral region, the saturation frequency may also be placed there.
STD-NMR. When a protein becomes saturated, ligands that are in exchange between the bound and the free form also become saturated when bound to the protein. By chemical exchange that saturation is carried into solution where it is detected. By subtraction of this spectrum from a spectrum without protein irradiation, one obtains an NMR spectrum that has only signals from molecules that bind to the protein. Nonbinders do not show up in the difference spectrum. The receptor protein is saturated with a selective saturation pulse. In general, the saturation pulse consists of a cascade of Gauss-shaped pulses. The duration of saturation times typically ranges from 1 to 2 s. The ligand is normally used in an approximately 50–100-fold molar excess over the protein, allowing one to work with low μM protein concentration
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Meyer, B. et al. (2004). Saturation Transfer Difference NMR Spectroscopy for Identifying Ligand Epitopes and Binding Specificities. In: Hamann, A., Asadullah, K., Schottelius, A. (eds) Leucocyte Trafficking. Ernst Schering Research Foundation Workshop, vol 44. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-05397-3_9
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DOI: https://doi.org/10.1007/978-3-662-05397-3_9
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