8B29
Human carbonic anhydrase II containing 6-fluorotryptophanes.
Summary for 8B29
| Entry DOI | 10.2210/pdb8b29/pdb |
| Descriptor | Carbonic anhydrase 2, ZINC ION (3 entities in total) |
| Functional Keywords | lyase |
| Biological source | Homo sapiens (human) |
| Total number of polymer chains | 1 |
| Total formula weight | 29262.14 |
| Authors | Pham, L.B.T.,Costantino, A.,Barbieri, L.,Calderone, V.,Luchinat, E.,Banci, L. (deposition date: 2022-09-13, release date: 2023-01-18, Last modification date: 2024-11-06) |
| Primary citation | Pham, L.B.T.,Costantino, A.,Barbieri, L.,Calderone, V.,Luchinat, E.,Banci, L. Direct Expression of Fluorinated Proteins in Human Cells for 19 F In-Cell NMR Spectroscopy. J.Am.Chem.Soc., 145:1389-1399, 2023 Cited by PubMed Abstract: In-cell NMR spectroscopy is a powerful approach to study protein structure and function in the native cellular environment. It provides precious insights into the folding, maturation, interactions, and ligand binding of important pharmacological targets directly in human cells. However, its widespread application is hampered by the fact that soluble globular proteins often interact with large cellular components, causing severe line broadening in conventional heteronuclear NMR experiments. F NMR can overcome this issue, as fluorine atoms incorporated in proteins can be detected by simple background-free 1D NMR spectra. Here, we show that fluorinated amino acids can be easily incorporated in proteins expressed in human cells by employing a medium switch strategy. This straightforward approach allows the incorporation of different fluorinated amino acids in the protein of interest, reaching fluorination efficiencies up to 60%, as confirmed by mass spectrometry and X-ray crystallography. The versatility of the approach is shown by performing F in-cell NMR on several proteins, including those that would otherwise be invisible by H-N in-cell NMR. We apply the approach to observe the interaction between an intracellular target, carbonic anhydrase 2, and its inhibitors, and to investigate how the formation of a complex between superoxide dismutase 1 and its chaperone CCS modulates the interaction of the chaperone subunit with the cellular environment. PubMed: 36604341DOI: 10.1021/jacs.2c12086 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.7 Å) |
Structure validation
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