Direct Expression of Fluorinated Proteins in Human Cells for 19F In-Cell NMR Spectroscopy
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 widespre...
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Published in | Journal of the American Chemical Society Vol. 145; no. 2; pp. 1389 - 1399 |
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Main Authors | , , , , , |
Format | Journal Article |
Language | English |
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American Chemical Society
18.01.2023
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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. 19F 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 19F in-cell NMR on several proteins, including those that would otherwise be invisible by 1H-15N 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. |
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AbstractList | 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. 19F 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 19F in-cell NMR on several proteins, including those that would otherwise be invisible by 1H-15N 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. 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. 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. |
Author | Calderone, Vito Banci, Lucia Barbieri, Letizia Costantino, Azzurra Pham, Lan B. T. Luchinat, Enrico |
AuthorAffiliation | Dipartimento di Chimica Dipartimento di Scienze e Tecnologie Agro-Alimentari Consorzio Interuniversitario Risonanze Magnetiche di Metallo ProteineCIRMMP Alma Mater StudiorumUniversità di Bologna Università degli Studi di Firenze CERMMagnetic Resonance Center |
AuthorAffiliation_xml | – name: Università degli Studi di Firenze – name: Dipartimento di Scienze e Tecnologie Agro-Alimentari – name: CERMMagnetic Resonance Center – name: Dipartimento di Chimica – name: Alma Mater StudiorumUniversità di Bologna – name: Consorzio Interuniversitario Risonanze Magnetiche di Metallo ProteineCIRMMP |
Author_xml | – sequence: 1 givenname: Lan B. T. surname: Pham fullname: Pham, Lan B. T. organization: Università degli Studi di Firenze – sequence: 2 givenname: Azzurra surname: Costantino fullname: Costantino, Azzurra organization: Università degli Studi di Firenze – sequence: 3 givenname: Letizia surname: Barbieri fullname: Barbieri, Letizia organization: Consorzio Interuniversitario Risonanze Magnetiche di Metallo ProteineCIRMMP – sequence: 4 givenname: Vito orcidid: 0000-0002-7963-6241 surname: Calderone fullname: Calderone, Vito organization: Università degli Studi di Firenze – sequence: 5 givenname: Enrico orcidid: 0000-0003-4183-4311 surname: Luchinat fullname: Luchinat, Enrico email: enrico.luchinat@unibo.it organization: Alma Mater StudiorumUniversità di Bologna – sequence: 6 givenname: Lucia orcidid: 0000-0003-0562-5774 surname: Banci fullname: Banci, Lucia email: banci@cerm.unifi.it organization: Università degli Studi di Firenze |
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Copyright | 2023 The Authors. Published by American Chemical Society 2023 The Authors. Published by American Chemical Society 2023 The Authors |
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Title | Direct Expression of Fluorinated Proteins in Human Cells for 19F In-Cell NMR Spectroscopy |
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