8b29
Human carbonic anhydrase II containing 6-fluorotryptophanes.Human carbonic anhydrase II containing 6-fluorotryptophanes.
Structural highlights
DiseaseCAH2_HUMAN Defects in CA2 are the cause of osteopetrosis autosomal recessive type 3 (OPTB3) [MIM:259730; also known as osteopetrosis with renal tubular acidosis, carbonic anhydrase II deficiency syndrome, Guibaud-Vainsel syndrome or marble brain disease. Osteopetrosis is a rare genetic disease characterized by abnormally dense bone, due to defective resorption of immature bone. The disorder occurs in two forms: a severe autosomal recessive form occurring in utero, infancy, or childhood, and a benign autosomal dominant form occurring in adolescence or adulthood. Autosomal recessive osteopetrosis is usually associated with normal or elevated amount of non-functional osteoclasts. OPTB3 is associated with renal tubular acidosis, cerebral calcification (marble brain disease) and in some cases with mental retardation.[1] [2] [3] [4] [5] FunctionCAH2_HUMAN Essential for bone resorption and osteoclast differentiation (By similarity). Reversible hydration of carbon dioxide. Can hydrate cyanamide to urea. Involved in the regulation of fluid secretion into the anterior chamber of the eye.[6] [7] Publication Abstract from PubMedIn-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. (19)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 (19)F in-cell NMR on several proteins, including those that would otherwise be invisible by (1)H-(15)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. Direct Expression of Fluorinated Proteins in Human Cells for (19)F In-Cell NMR Spectroscopy.,Pham LBT, Costantino A, Barbieri L, Calderone V, Luchinat E, Banci L J Am Chem Soc. 2023 Jan 18;145(2):1389-1399. doi: 10.1021/jacs.2c12086. Epub 2023 , Jan 5. PMID:36604341[8] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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