8B29

Human carbonic anhydrase II containing 6-fluorotryptophanes.

8B29 の概要

• エントリーDOI: 10.2210/pdb8b29/pdb
• 分子名称: Carbonic anhydrase 2, ZINC ION (3 entities in total)
• 機能のキーワード: lyase
• 由来する生物種: Homo sapiens (human)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 29262.14

構造登録者

Pham, L.B.T.,Costantino, A.,Barbieri, L.,Calderone, V.,Luchinat, E.,Banci, L. (登録日: 2022-09-13, 公開日: 2023-01-18, 最終更新日: 2024-11-06)

主引用文献

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

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: 36604341
DOI: 10.1021/jacs.2c12086

実験手法

X-RAY DIFFRACTION (1.7 Å)