Direct Expression of Fluorinated Proteins in Human Cells for 19 F 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...
Saved in:
Published in | Journal of the American Chemical Society Vol. 145; no. 2; pp. 1389 - 1399 |
---|---|
Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
18.01.2023
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
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. |
---|---|
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.
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. |
Author | Calderone, Vito Banci, Lucia Barbieri, Letizia Costantino, Azzurra Pham, Lan B T Luchinat, Enrico |
Author_xml | – sequence: 1 givenname: Lan B T surname: Pham fullname: Pham, Lan B T organization: CERM─Magnetic Resonance Center, Università degli Studi di Firenze, Via Luigi Sacconi 6, 50019Sesto Fiorentino, Italy – sequence: 2 givenname: Azzurra surname: Costantino fullname: Costantino, Azzurra organization: CERM─Magnetic Resonance Center, Università degli Studi di Firenze, Via Luigi Sacconi 6, 50019Sesto Fiorentino, Italy – sequence: 3 givenname: Letizia surname: Barbieri fullname: Barbieri, Letizia organization: Consorzio Interuniversitario Risonanze Magnetiche di Metallo Proteine─CIRMMP, Via Luigi Sacconi 6, 50019Sesto Fiorentino, Italy – sequence: 4 givenname: Vito orcidid: 0000-0002-7963-6241 surname: Calderone fullname: Calderone, Vito organization: Dipartimento di Chimica, Università degli Studi di Firenze, Via della Lastruccia 3, 50019Sesto Fiorentino, Italy – sequence: 5 givenname: Enrico orcidid: 0000-0003-4183-4311 surname: Luchinat fullname: Luchinat, Enrico organization: Dipartimento di Scienze e Tecnologie Agro-Alimentari, Alma Mater Studiorum─Università di Bologna, Piazza Goidanich 60, 47521Cesena, Italy – sequence: 6 givenname: Lucia orcidid: 0000-0003-0562-5774 surname: Banci fullname: Banci, Lucia organization: Dipartimento di Chimica, Università degli Studi di Firenze, Via della Lastruccia 3, 50019Sesto Fiorentino, Italy |
BackLink | https://www.ncbi.nlm.nih.gov/pubmed/36604341$$D View this record in MEDLINE/PubMed |
BookMark | eNo9kMtqwzAQRUVJaR7truuiD6jTGcmW5WVJkyaQPuhj042RZQkcHMlICTR_X4e0XQ13ONwLZ0wGzjtDyDXCFIHh3UbpOGUaGUhxRkaYMUgyZGJARgDAklwKPiTjGDd9TJnECzLkQkDKUxyRr4cmGL2j8-8umBgb76i3dNHufWic2pmavga_M42LtHF0ud8qR2embSO1PlAs6IKuXHL80OenN_re9WXBR-27wyU5t6qN5ur3TsjnYv4xWybrl8fV7H6daASOidBFjiytMi4zXaFAaYHnkhtlodY8q3RhNNaZ5FAUoKCocmN1DqlRTFvM-YTcnnp1PxyDsWUXmq0KhxKhPCoqj4rKX0U9fnPCu321NfU__OeE_wBmz2Lo |
CitedBy_id | crossref_primary_10_1021_acscentsci_3c00385 crossref_primary_10_1016_j_pnmrs_2023_10_002 crossref_primary_10_1360_SSC_2023_0247 crossref_primary_10_1039_D4SC01755B crossref_primary_10_1002_chem_202303988 crossref_primary_10_1002_pro_4910 crossref_primary_10_1002_pro_4950 crossref_primary_10_1002_anie_202300318 crossref_primary_10_1002_cbic_202400195 crossref_primary_10_1002_chem_202400323 crossref_primary_10_1021_jacsau_3c00712 crossref_primary_10_1002_ange_202300318 crossref_primary_10_1007_s12210_023_01168_y crossref_primary_10_1016_j_jinorgbio_2023_112236 crossref_primary_10_1039_D3SC00519D crossref_primary_10_1038_s41598_023_49247_2 |
Cites_doi | 10.1002/prot.340040406 10.1021/acs.accounts.8b00147 10.1021/acs.chemrev.1c00937 10.1002/anie.202201097 10.1039/c2cc35347d 10.1021/acs.chemrev.1c00790 10.1038/nature07839 10.1016/j.celrep.2020.108074 10.1002/anie.201207243 10.1021/acs.jmedchem.5b01447 10.1107/S0907444909042073 10.1021/jacs.1c10104 10.1107/S2059798321009037 10.1002/anie.201900840 10.1021/acs.analchem.0c01677 10.1073/pnas.2019918118 10.1038/s41598-017-17815-y 10.1038/nchembio.1202 10.3390/ijms22116042 10.1002/anie.202007184 10.1007/s10858-021-00358-w 10.1093/nar/gkx109 10.1021/ja904407w 10.1107/S0907444906029799 10.1021/jacs.8b08436 10.1107/S0907444909042589 10.1073/pnas.1518620113 10.1002/anie.201913436 10.1107/S0907444910007493 10.1107/S0907444909047337 10.1021/acschembio.7b00879 10.1038/nprot.2016.061 10.1016/j.pep.2004.06.031 10.1002/anie.201500261 10.1021/bi900872p 10.1038/nature16531 10.1096/fj.201900474R 10.1021/acs.biochem.7b00938 10.1007/s00775-017-1509-5 10.1074/jbc.M411805200 10.1038/s41592-019-0334-x 10.1021/ja907966n 10.1021/ja064661t 10.1107/S0907444909052925 |
ContentType | Journal Article |
DBID | CGR CUY CVF ECM EIF NPM AAYXX CITATION |
DOI | 10.1021/jacs.2c12086 |
DatabaseName | Medline MEDLINE MEDLINE (Ovid) MEDLINE MEDLINE PubMed CrossRef |
DatabaseTitle | MEDLINE Medline Complete MEDLINE with Full Text PubMed MEDLINE (Ovid) CrossRef |
DatabaseTitleList | MEDLINE |
Database_xml | – sequence: 1 dbid: NPM name: PubMed url: https://proxy.k.utb.cz/login?url=http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed sourceTypes: Index Database – sequence: 2 dbid: EIF name: MEDLINE url: https://proxy.k.utb.cz/login?url=https://www.webofscience.com/wos/medline/basic-search sourceTypes: Index Database |
DeliveryMethod | fulltext_linktorsrc |
Discipline | Chemistry |
EISSN | 1520-5126 |
EndPage | 1399 |
ExternalDocumentID | 10_1021_jacs_2c12086 36604341 |
Genre | Research Support, Non-U.S. Gov't Journal Article |
GroupedDBID | --- -DZ -ET -~X .DC .K2 4.4 53G 55A 5GY 5RE 5VS 7~N 85S AABXI AAHBH ABJNI ABMVS ABPPZ ABQRX ABUCX ACBEA ACGFO ACGFS ACJ ACNCT ACS ADHLV AEESW AENEX AFEFF AGXLV AHGAQ ALMA_UNASSIGNED_HOLDINGS AQSVZ BAANH BKOMP CGR CS3 CUPRZ CUY CVF DU5 EBS ECM ED~ EIF F5P GGK GNL IH2 IH9 JG~ LG6 NPM P2P ROL RXW TAE TN5 UHB UI2 UKR UPT VF5 VG9 VQA W1F WH7 XSW YQT YZZ ZCA ~02 AAYXX CITATION |
ID | FETCH-LOGICAL-c1031-6c97124b5385cb1618f03783eaf0dc35bc9ec1d5830990a09b7efc704ea2cf173 |
IEDL.DBID | ACS |
ISSN | 0002-7863 |
IngestDate | Fri Aug 23 00:58:24 EDT 2024 Sat Sep 28 08:15:05 EDT 2024 |
IsPeerReviewed | true |
IsScholarly | true |
Issue | 2 |
Language | English |
LinkModel | DirectLink |
MergedId | FETCHMERGED-LOGICAL-c1031-6c97124b5385cb1618f03783eaf0dc35bc9ec1d5830990a09b7efc704ea2cf173 |
ORCID | 0000-0003-4183-4311 0000-0003-0562-5774 0000-0002-7963-6241 |
PMID | 36604341 |
PageCount | 11 |
ParticipantIDs | crossref_primary_10_1021_jacs_2c12086 pubmed_primary_36604341 |
PublicationCentury | 2000 |
PublicationDate | 2023-01-18 |
PublicationDateYYYYMMDD | 2023-01-18 |
PublicationDate_xml | – month: 01 year: 2023 text: 2023-01-18 day: 18 |
PublicationDecade | 2020 |
PublicationPlace | United States |
PublicationPlace_xml | – name: United States |
PublicationTitle | Journal of the American Chemical Society |
PublicationTitleAlternate | J Am Chem Soc |
PublicationYear | 2023 |
References | ref9/cit9 ref45/cit45 ref6/cit6 ref36/cit36 ref3/cit3 ref27/cit27 ref18/cit18 ref11/cit11 ref25/cit25 ref16/cit16 ref29/cit29 ref32/cit32 ref23/cit23 ref39/cit39 ref14/cit14 ref8/cit8 ref5/cit5 ref31/cit31 ref2/cit2 ref43/cit43 ref34/cit34 ref37/cit37 ref28/cit28 ref40/cit40 ref20/cit20 ref17/cit17 ref10/cit10 ref26/cit26 ref35/cit35 ref19/cit19 ref21/cit21 ref12/cit12 Abràmoff M. D. (ref33/cit33) 2004; 11 ref15/cit15 ref42/cit42 ref41/cit41 ref22/cit22 ref13/cit13 ref4/cit4 ref30/cit30 ref1/cit1 ref24/cit24 ref38/cit38 ref44/cit44 ref7/cit7 |
References_xml | – ident: ref37/cit37 doi: 10.1002/prot.340040406 – ident: ref27/cit27 doi: 10.1021/acs.accounts.8b00147 – ident: ref2/cit2 doi: 10.1021/acs.chemrev.1c00937 – ident: ref23/cit23 doi: 10.1002/anie.202201097 – ident: ref24/cit24 doi: 10.1039/c2cc35347d – ident: ref1/cit1 doi: 10.1021/acs.chemrev.1c00790 – ident: ref25/cit25 doi: 10.1038/nature07839 – ident: ref7/cit7 doi: 10.1016/j.celrep.2020.108074 – ident: ref12/cit12 doi: 10.1002/anie.201207243 – ident: ref15/cit15 doi: 10.1021/acs.jmedchem.5b01447 – ident: ref41/cit41 doi: 10.1107/S0907444909042073 – ident: ref45/cit45 doi: 10.1021/jacs.1c10104 – ident: ref29/cit29 doi: 10.1107/S2059798321009037 – ident: ref6/cit6 doi: 10.1002/anie.201900840 – ident: ref14/cit14 doi: 10.1021/acs.analchem.0c01677 – ident: ref17/cit17 doi: 10.1073/pnas.2019918118 – ident: ref30/cit30 doi: 10.1038/s41598-017-17815-y – ident: ref3/cit3 doi: 10.1038/nchembio.1202 – ident: ref22/cit22 doi: 10.3390/ijms22116042 – ident: ref8/cit8 doi: 10.1002/anie.202007184 – ident: ref11/cit11 doi: 10.1007/s10858-021-00358-w – ident: ref20/cit20 doi: 10.1093/nar/gkx109 – ident: ref26/cit26 doi: 10.1021/ja904407w – ident: ref32/cit32 doi: 10.1107/S0907444906029799 – ident: ref21/cit21 doi: 10.1021/jacs.8b08436 – ident: ref38/cit38 doi: 10.1107/S0907444909042589 – ident: ref4/cit4 doi: 10.1073/pnas.1518620113 – ident: ref10/cit10 doi: 10.1002/anie.201913436 – ident: ref40/cit40 doi: 10.1107/S0907444910007493 – volume: 11 start-page: 36 year: 2004 ident: ref33/cit33 publication-title: Biophotonics Int. contributor: fullname: Abràmoff M. D. – ident: ref36/cit36 doi: 10.1107/S0907444909047337 – ident: ref9/cit9 doi: 10.1021/acschembio.7b00879 – ident: ref28/cit28 doi: 10.1038/nprot.2016.061 – ident: ref35/cit35 doi: 10.1016/j.pep.2004.06.031 – ident: ref18/cit18 doi: 10.1002/anie.201500261 – ident: ref43/cit43 doi: 10.1021/bi900872p – ident: ref5/cit5 doi: 10.1038/nature16531 – ident: ref34/cit34 doi: 10.1096/fj.201900474R – ident: ref13/cit13 doi: 10.1021/acs.biochem.7b00938 – ident: ref31/cit31 doi: 10.1007/s00775-017-1509-5 – ident: ref42/cit42 doi: 10.1074/jbc.M411805200 – ident: ref44/cit44 doi: 10.1038/s41592-019-0334-x – ident: ref16/cit16 doi: 10.1021/ja907966n – ident: ref19/cit19 doi: 10.1021/ja064661t – ident: ref39/cit39 doi: 10.1107/S0907444909052925 |
SSID | ssj0004281 |
Score | 2.461838 |
Snippet | In-cell NMR spectroscopy is a powerful approach to study protein structure and function in the native cellular environment. It provides precious insights into... |
SourceID | crossref pubmed |
SourceType | Aggregation Database Index Database |
StartPage | 1389 |
SubjectTerms | Amino Acids Fluorine - chemistry Humans Magnetic Resonance Spectroscopy - methods Molecular Chaperones Nuclear Magnetic Resonance, Biomolecular - methods |
Title | Direct Expression of Fluorinated Proteins in Human Cells for 19 F In-Cell NMR Spectroscopy |
URI | https://www.ncbi.nlm.nih.gov/pubmed/36604341 |
Volume | 145 |
hasFullText | 1 |
inHoldings | 1 |
isFullTextHit | |
isPrint | |
link | http://utb.summon.serialssolutions.com/2.0.0/link/0/eLvHCXMwjV1LS8NAEF7Ei158P-qLOehxS5JNspujhMYqtIhaKF5KdrMLxZIWm4L6693Jw9J60GtIQpgN83278803hFwbbM7k2tDIz3yKlJoKg8oc15NYSJO6tC_u9cPuwH8YBsOlQHa9gu-hP5Catz3lepZ8Y6q1kIYMKH5etj96wm1YLhchq_Xt6w-vIM8KhyyxJNkld01HTiUheWsvCtlWX78NGv_4zD2yU9NJuK3Wf59s6PyAbMXNFLdD8lrlNOh81IrXHKYGkskChXeWZ2bwiE4N43wO4xzKI32I9WQyB8tmwY0ggfuc4hXo954Ax9UXaIA5nX0ekUHSeYm7tJ6nQBUOc6ChiriFc2lzXKAkOuUbh3HBdGqcTLFAqkgrNwsEw2pZ6kTSLqLijq9TTxmXs2OymU9zfUpApExnjFnuYPdTmfIFYxbsOVMs1FKkUYvcNLEezSrbjFFZ7vbsdsNGalRHqkVOqoX4uYuFoeNbaD375xvOyTZOgMdTEVdckM3ifaEvLU8o5FX5l3wDgNi1dg |
link.rule.ids | 315,786,790,2782,27957,27958 |
linkProvider | American Chemical Society |
openUrl | ctx_ver=Z39.88-2004&ctx_enc=info%3Aofi%2Fenc%3AUTF-8&rfr_id=info%3Asid%2Fsummon.serialssolutions.com&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Ajournal&rft.genre=article&rft.atitle=Direct+Expression+of+Fluorinated+Proteins+in+Human+Cells+for+19+F+In-Cell+NMR+Spectroscopy&rft.jtitle=Journal+of+the+American+Chemical+Society&rft.au=Pham%2C+Lan+B+T&rft.au=Costantino%2C+Azzurra&rft.au=Barbieri%2C+Letizia&rft.au=Calderone%2C+Vito&rft.date=2023-01-18&rft.eissn=1520-5126&rft.volume=145&rft.issue=2&rft.spage=1389&rft_id=info:doi/10.1021%2Fjacs.2c12086&rft_id=info%3Apmid%2F36604341&rft.externalDocID=36604341 |
thumbnail_l | http://covers-cdn.summon.serialssolutions.com/index.aspx?isbn=/lc.gif&issn=0002-7863&client=summon |
thumbnail_m | http://covers-cdn.summon.serialssolutions.com/index.aspx?isbn=/mc.gif&issn=0002-7863&client=summon |
thumbnail_s | http://covers-cdn.summon.serialssolutions.com/index.aspx?isbn=/sc.gif&issn=0002-7863&client=summon |