Binding Moiety Mapping by Saturation Transfer Difference NMR

Abstract

Saturation transfer difference (STD) NMR has emerged as one of the key technologies in lead optimization during drug design. Unlike most biophysical assays which report only on the binding affinity, STD NMR reports simultaneously on both the binding affinity and the structure of the binding ligand/protein complex. The STD experiment drives magnetization from a protein to a bound small molecule ligand which carries away the memory of the saturation signal when it dissociates. Since the transfer of saturation is distance dependent, STD NMR can be used to map the specific atoms on the ligand in contact with a protein receptor allowing the impact of any structural change in the binding site to be mapped directly on to the individual functional groups responsible when a suitable compound library is screened. Because the signal is detected from the free ligand and not the bound complex, it can be used on a much wider range of systems than protein-detected NMR and has the advantage of more directly reporting on distances than changes in chemical shifts alone. The STD experiment, while deceptively simple, is very sensitive to both sample conditions and acquisition parameters. We present a general protocol for setting up and STD NMR experiment with a particular focus on how choices in sample conditions and acquisition parameters affect the outcome of the experiment.

New Insights into the Binding Site and Affinity of the Interaction between Biotin and PAMAMs-NH2 via NMR Studies

“…Saturation transfer difference (STD) spectroscopy is used to determine the interaction between the host and the guests. − The STD experiments drive magnetization through the entire host and eventually to any bound ligands . Saturation transfer double difference (STDD) NMR spectra can be obtained after subtraction the one of host molecule from the whole host–guest complex STD spectrum, resulting in the disappearance of strong background and overlapping signals from host molecule. − Since the transfer of saturation magnetization is distance dependent, the STDD method can also be used to map the interaction intensity degree of different protons of the guest with the host. ,− On the basis of the methods, the mechanism of the host–guest interaction can be deduced.…”

Elucidation of the Binding Orientation in α2,3- and α2,6-Linked Neu5Ac-Gal Epitopes toward a Hydrophilic Molecularly Imprinted Monolith

“…However, knowledge of what parts of the target molecule actually contribute to the binding is equally interesting in the design of MIPs. A powerful tool for simultaneous study of reversible binding affinity and ligand-substrate interactions at the molecular level is saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, − which is based on the distance- and time-dependent transfer of saturation from protons of a saturated substrate to those of the ligand by the intermolecular nuclear Overhauser effect (NOE) . This allows investigations of analyte binding toward their corresponding MIPs under realistic experimental conditions, , as well as mapping of epitopes bound to affinity resins. − In these applications, STD-NMR is used to identify the binding epitopes of low molecular weight ligands by mapping the protons that are in close contact with the substrate when the complex is formed. , Protons of the released ligands that have been situated in the closest proximity to the receptor during a binding event show the highest STD effects and vice versa.…”