Issue link: https://resources.nanotempertech.com/i/1133236
8 Nuclear Magnetic Resonance Nuclear Magnetic Resonance (NMR) has become not only a valuable screening tool for analyzing the binding of ligands to protein targets, but it also provides structural information on protein–ligand interactions used in the optimization of weak-binding hits into high-affinity leads. NMR has two main approaches: protein-observed, where changes are detected in the spectrum of the protein, and ligand- observed, where changes are detected in the ligand's spectrum. Heteronuclear single-quantum coherence (HSQC) is the main technique in protein-observed NMR. Transfer of signal between labeled atoms from hydrogen to either nitrogen or carbon results in spectra that shows a single peak where each amide or methyl group resides. The position of the peaks is impacted by changes to the local chemical environment (chemical shi ) resulting from ligand binding events. The dynamic range of HQSC is wide, typically limited only by fragment solubility. Information confirming that different fragments are binding to the same site can be obtained. If the spectrum of the protein can be assigned, the location of the binding site can also be determined. A K d can be obtained from the correlation curve between chemical shi changes and ligand concentrations. Protein-observed NMR has several limitations. Proteins must be smaller than 35 kDa, soluble within 20 to 100 µM and isotopically labeled. In addition, relatively large amounts of target protein are required. For proteins over 35 kDa, the only NMR option is ligand-observed. In this approach, ligand is used at 50 times the concentration of protein (usually about 10 µM) to generate ligand-observed spectra. The major techniques are saturation transfer difference (STD), water ligand-observed via gradient spectroscopy (water-LOGSY), and Carr- Purcell-Meiboom-Gill (CPMG). Great for obtaining K d and if protein observed, also structural information Limited by large amount of sample needed (protein observed), costly equipment and medium throughput