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Direct Expression of Fluorinated Proteins in Human Cells for 19F In-Cell NMR Spectroscopy.
| Content Provider | Europe PMC |
|---|---|
| Author | Pham, Lan B.T. Costantino, Azzurra Barbieri, Letizia Calderone, Vito Luchinat, Enrico Banci, Lucia |
| Copyright Year | 2023 |
| Abstract | In-cell NMR spectroscopyis a powerful approach to study proteinstructure and function in the native cellular environment. It providesprecious insights into the folding, maturation, interactions, andligand binding of important pharmacological targets directly in humancells. However, its widespread application is hampered by the factthat soluble globular proteins often interact with large cellularcomponents, causing severe line broadening in conventional heteronuclearNMR experiments. 19F NMR can overcome this issue, as fluorineatoms incorporated in proteins can be detected by simple background-free1D NMR spectra. Here, we show that fluorinated amino acids can beeasily incorporated in proteins expressed in human cells by employinga medium switch strategy. This straightforward approach allows theincorporation of different fluorinated amino acids in the proteinof interest, reaching fluorination efficiencies up to 60%, as confirmedby mass spectrometry and X-ray crystallography. The versatility ofthe approach is shown by performing 19F in-cell NMR onseveral proteins, including those that would otherwise be invisibleby 1H-15N in-cell NMR. We apply the approachto observe the interaction between an intracellular target, carbonicanhydrase 2, and its inhibitors, and to investigate how the formationof a complex between superoxide dismutase 1 and its chaperone CCSmodulates the interaction of the chaperone subunit with the cellularenvironment. |
| ISSN | 00027863 |
| Volume Number | 145 |
| PubMed Central reference number | PMC9853860 |
| Issue Number | 2 |
| PubMed reference number | 36604341 |
| Journal | Journal of the American Chemical Society [J. Am. Chem. Soc] |
| e-ISSN | 15205126 |
| DOI | 10.1021/jacs.2c12086 |
| Language | English |
| Publisher | American Chemical Society |
| Publisher Date | 2023-01-05 |
| Access Restriction | Open |
| Rights License | Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained (https://creativecommons.org/licenses/by/4.0/). © 2023 The Authors. Published by American Chemical Society |
| Content Type | Text |
| Resource Type | Article |
| Subject | Chemistry Colloid and Surface Chemistry Biochemistry Catalysis |
