Saturation Transfer Difference Spectroscopy

Abstract

Saturation transfer difference (STD) NMR is a powerful technique for detection and characterization of reversible binding interactions between small molecule ligands and macromolecular receptors. The STD methodology is broadly used for detection of protein–ligand interactions and for fragment screening and has also been successfully applied to complex systems such as reconstituted membranes, intact cells, platelets, or whole viruses. The basic STD experiment relies on1H NMR (Nuclear Magnetic Resonance) difference spectroscopy. Saturation transferred from the receptor to bound ligands in the complex is measured by observing the NMR signal of the ligand. The corresponding STD NMR spectrum reveals only signals of binding compounds. Quantitative analysis of the STD effects allows the identification of the binding epitope of a ligand when bound to its receptor and can also provide affinity information. Water–ligand observed gradient spectroscopy (WaterLOGSY) is a related method for the characterization of transient ligand–receptor interactions, but unlike STD, in WaterLOGSY experiments, the bulk water is the origin of the perturbation transfer.

Anisotropic 1H STD‐NMR Spectroscopy: Exploration of Enantiomer‐Polypeptide Interactions in Chiral Oriented Environments

“…This is driven by the duration of polymer saturation, t sat , as well as the exchange features between "bound" and "free" states. [28,29,37] This latter corresponds to the time-residence, t res , of the transient polymer-solute complex and fractions of solutes in both states as well (see Figure 1a).…”

“…The basic principle of 1 H STD-NMR in anisotropic media is formally identical to that applied to isotropic samples. [27][28][29][30][31] Hence, from a practical point of view, the anisotropic STD-NMR experiments can be performed using the routine STD-NMR pulse sequence developed in isotropic liquids, with no peculiar modification (see Figure SI-2).…”

“…Indeed, in the first approach explored previously, there are several biases that may prevent a direct and reliable interpretation of STD signals in terms of internuclei distances and thus of relevant geometrical insights on the binding process. Possible biases are: i) a fast polymer-solute rebinding process during t sat , [28,[46][47][48] and ii) a strong variation of longitudinal relaxation time, T 1 ( 1 H), from siteto-site. [46][47][48] While the former is here negligible owing to the high solute-to-polymer ratio used in this work, the latter has to be considered, given the dispersion of T 1 ( 1 H) at the atomic level (see Section VII in SI) and because t sat is here higher than T 1 ( 1 H) values.…”

“…[26] To this aim, we propose to characterize directly the oriented transient polymer-analyte array by saturation transfer difference (STD) NMR spectroscopy. [27,28] This is a well-established method to probe and characterize small molecules in weak interaction with a macromolecule based on intermolecular magnetization transfers. It has been widely used to assess ligand-protein interactions both in terms of affinity (determination of dissociation constants K D ) and geometry (epitope mapping).…”

“…It has been widely used to assess ligand-protein interactions both in terms of affinity (determination of dissociation constants K D ) and geometry (epitope mapping). [27][28][29][30][31] Interestingly, this tool has also been employed for investigating polar solutes interacting with soft matters (as stationary phases used in HPLC) by 1 H high-resolution magic angle spinning (HRMAS) NMR at low speed, [32] as well as applied successfully to characterize enantiomers-macromolecule interactions in achiral isotropic liquids. [33][34][35][36] However, the STD-NMR approach has never been tested so far to investigate the case of chiral lyotropic liquid crystals, to the best of our knowledge.…”