Solution NMR spectroscopy has been used in various research topics, including protein–protein, protein–nucleotide complexes, and membrane proteins, to provide useful information in order to understand protein structure and function. Although it is still challenging to study protein structures with high molecular mass due to the signal overlap and sensitivity, NMR has been widely used in protein chemistry and drug discovery with the development of magnets, pulse programs, and different protein-labeling strategies. Solution NMR spectroscopy is able to investigate protein structures and dynamics under solution conditions because the targets can be studied in different buffers and at various temperatures. Other methods, such as small-angle X-ray/neutron scattering (SAXS/SANS), mass spectrometry and chemical cross-linking are also used to determine structures of protein complexes. For example, the structures of many difficult targets such as ion channels and membrane-bound enzyme complexes were obtained using cryo-EM. In recent years, the rapid development of cryo-EM has made it possible to solve structures of biomolecule complexes with high molecular weight at a high resolution. When diffracted crystals are available, X-ray crystallography is a robust way to obtain high-resolution structures of biomolecules. Solution nuclear magnetic resonance (NMR), X-ray crystallography and cryogenic electron microscopy (cryo-EM) are important tools for obtaining the structures of biomolecules at atomic resolution. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement. In this review, applications of in-cell NMR are summarized. Despite the challenges in this method, progress has been made in recent years. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. ![]() Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions in living cells.
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