{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T14:07:26Z","timestamp":1789567646356,"version":"build-2803163510"},"reference-count":22,"publisher":"American Chemical Society (ACS)","issue":"36","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["5P30GM110758-02"],"award-info":[{"award-number":["5P30GM110758-02"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Institutes of Health (NIH)","award":["1S10OD025185-01A1"],"award-info":[{"award-number":["1S10OD025185-01A1"]}]},{"name":"National Institutes of Health (NIH)","award":["1U54AI170791"],"award-info":[{"award-number":["1U54AI170791"]}]},{"name":"National Science Foundation (NSF)","award":["CHE0959496"],"award-info":[{"award-number":["CHE0959496"]}]},{"name":"National Science Foundation (NSF)","award":["MCB2231099"],"award-info":[{"award-number":["MCB2231099"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,16]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy is uniquely positioned to yield atomic-level structural and dynamic information on large proteins and protein assemblies in cellular environments. Yet, in-cell MAS NMR applications using the most common biomolecular reporter nuclei-1H, 13C, and 15N-remain hampered by a large cellular background, inherently low sensitivity, and limited resolution. In contrast, 19F is a high-receptivity nucleus that is naturally absent in biological molecules and is an excellent probe for in-cell MAS NMR. Here, we report a 19F-labeling strategy that combines site-specific incorporation of 4-(trifluoromethyl)-l-phenylalanine (tfmF) via genetic code expansion with fluorination of all tryptophan residues at the 7 position of the indole ring. We apply this approach to the N-terminal domain of the nucleocapsid protein from SARS-CoV-2, NNTD. For a microcrystalline 7F-Trp,tfmF28,U\u201315N\u2013NNTD sample, 19F\u201319F correlations between the CF3 group of the tfmF and the F atoms of the three 7F-Trp residues are detected in 2D dipolar-based experiments, spanning a distance range from 10 to 21 \u00c5. Additionally, for 7F-Trp,tfmF28,U\u201315N\u2013NNTD electroporated into A2780 cells, the increased spectral resolution and sensitivity afforded by the incorporation of the CF3 group enable the acquisition of high-quality 19F in-cell MAS NMR spectra within a few hours. The simultaneous labeling with an aromatic fluorine atom and a CF3 group expands the toolbox of 19F MAS NMR for cellular structural biology.<\/jats:p>","DOI":"10.1021\/jacs.6c10748","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T10:50:19Z","timestamp":1788432619000},"page":"38924-38931","source":"Crossref","is-referenced-by-count":0,"title":["Simultaneous\nSite- and Residue-Specific Incorporation\nof Fluorinated Amino Acids for In-Cell Structural Biology by 19F Magic Angle Spinning NMR Spectroscopy"],"prefix":"10.1021","volume":"148","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8636-0087","authenticated-orcid":true,"given":"Kumar","family":"Tekwani Movellan","sequence":"first","affiliation":[{"name":"University of Delaware , , , ,","place":["Newark, Delaware, United States, 19716"]},{"name":"University of Pittsburgh School of Medicine , , 1051 Biomedical Science Tower 3, 3501 Fifth Avenue , , ,","place":["Pittsburgh, Pennsylvania, United States, 15261"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9072-3525","authenticated-orcid":true,"given":"Angela M.","family":"Gronenborn","sequence":"additional","affiliation":[{"name":"University of Delaware , , , ,","place":["Newark, Delaware, United States, 19716"]},{"name":"University of Pittsburgh School of Medicine , , 1051 Biomedical Science Tower 3, 3501 Fifth Avenue , , ,","place":["Pittsburgh, Pennsylvania, United States, 15261"]},{"name":"University of Pittsburgh School of Medicine , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15261"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0346-1131","authenticated-orcid":true,"given":"Tatyana","family":"Polenova","sequence":"additional","affiliation":[{"name":"University of Delaware , , , ,","place":["Newark, Delaware, United States, 19716"]},{"name":"University of Pittsburgh School of Medicine , , 1051 Biomedical Science Tower 3, 3501 Fifth Avenue , , ,","place":["Pittsburgh, Pennsylvania, United States, 15261"]},{"name":"University of Pittsburgh School of Medicine , , , ,","place":["Pittsburgh, Pennsylvania, United States, 15261"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,1]]},"reference":[{"issue":"23","key":"2026091609105854900_cit1","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202201097","article-title":"Visualizing\nproteins in mammalian cells by 19F NMR spectroscopy","volume":"61","author":"Zhu","year":"2022","journal-title":"Angew. Chem., Int. Ed."},{"issue":"2","key":"2026091609105854900_cit2","doi-asserted-by":"publisher","first-page":"1389","DOI":"10.1021\/jacs.2c12086","article-title":"Direct expression of fluorinated\nproteins in human cells for 19F in-cell NMR spectroscopy","volume":"145","author":"Pham","year":"2023","journal-title":"J. Am. Chem. Soc."},{"issue":"7","key":"2026091609105854900_cit3","doi-asserted-by":"publisher","first-page":"6744","DOI":"10.1021\/jacs.5c18349","article-title":"Endogenous site-specific encoding\nof trifluoromethyl-bearing phenylalanine and tryptophan for in-cell 19F NMR","volume":"148","author":"Augustin","year":"2026","journal-title":"J. Am. Chem. Soc."},{"issue":"40","key":"2026091609105854900_cit4","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.adq3115","article-title":"Expanding the tool box for native structural biology: 19F dynamic nuclear polarization with fast magic angle spinning","volume":"10","author":"Movellan","year":"2024","journal-title":"Sci. Adv."},{"issue":"33","key":"2026091609105854900_cit5","doi-asserted-by":"publisher","first-page":"29970","DOI":"10.1021\/jacs.5c06739","article-title":"Pushing sensitivity and specificity limits in native\nstructural biology: 19F multinuclear dynamic nuclear polarization\nwith magic angle spinning","volume":"147","author":"Movellan","year":"2025","journal-title":"J. Am. Chem. Soc."},{"issue":"5","key":"2026091609105854900_cit6","doi-asserted-by":"publisher","first-page":"1160","DOI":"10.1021\/ja064661t","article-title":"Site-specific\nincorporation of a 19F-amino acid into proteins as an NMR\nprobe for characterizing\nprotein structure and reactivity","volume":"129","author":"Jackson","year":"2007","journal-title":"J. Am. Chem.\nSoc."},{"issue":"7","key":"2026091609105854900_cit7","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202419709","article-title":"High-efficiency\ntrifluoromethyl-methionine incorporation into cyclophilin A by cell-free\nsynthesis for 19F NMR studies","volume":"64","author":"Zhu","year":"2025","journal-title":"Angew.\nChem., Int. Ed."},{"issue":"12","key":"2026091609105854900_cit8","doi-asserted-by":"publisher","first-page":"3601","DOI":"10.1111\/febs.70446","article-title":"Comparative\nevaluation of noncanonical amino acids as site-specific NMR probes\nfor the complex of E. coli SSB with single-stranded\nDNA without isotope labelling","volume":"293","author":"Tharayil","year":"2026","journal-title":"FEBS J."},{"issue":"25","key":"2026091609105854900_cit9","doi-asserted-by":"publisher","first-page":"5953","DOI":"10.1021\/bi900426d","article-title":"Probing protein folding using site-specifically\nencoded unnatural amino acids as FRET donors with tryptophan","volume":"48","author":"Miyake-Stoner","year":"2009","journal-title":"Biochemistry"},{"issue":"7588","key":"2026091609105854900_cit10","doi-asserted-by":"publisher","first-page":"45","DOI":"10.1038\/nature16531","article-title":"Structural disorder\nof monomeric \u03b1-synuclein persists in mammalian cells","volume":"530","author":"Theillet","year":"2016","journal-title":"Nature"},{"issue":"23","key":"2026091609105854900_cit11","doi-asserted-by":"publisher","first-page":"10543","DOI":"10.1021\/jacs.2c03320","article-title":"Atomic-resolution structure of SARS-CoV-2\nnucleocapsid protein N-terminal domain","volume":"144","author":"Sarkar","year":"2022","journal-title":"J. Am.\nChem. Soc."},{"issue":"1","key":"2026091609105854900_cit12","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1016\/j.jmr.2008.09.019","article-title":"Measurement of sample temperatures under magic-angle spinning from\nthe chemical shift and spin-lattice relaxation rate of 79Br in KBr powder","volume":"196","author":"Thurber","year":"2009","journal-title":"J. Magn. Reson."},{"key":"2026091609105854900_cit13","doi-asserted-by":"publisher","first-page":"18","DOI":"10.1016\/j.jmr.2013.04.009","article-title":"Broadband\nhomonuclear correlation spectroscopy driven by combined R2nv sequences under fast magic angle spinning for NMR structural\nanalysis of organic and biological solids","volume":"232","author":"Hou","year":"2013","journal-title":"J.\nMagn. Reson."},{"issue":"23","key":"2026091609105854900_cit14","doi-asserted-by":"publisher","first-page":"8210","DOI":"10.1021\/acs.analchem.1c00784","article-title":"Fast 19F magic angle spinning\nNMR crystallography\nfor structural characterization of fluorine-containing pharmaceutical\ncompounds","volume":"93","author":"Guo","year":"2021","journal-title":"Anal. Chem."},{"issue":"38","key":"2026091609105854900_cit15","doi-asserted-by":"publisher","first-page":"13029","DOI":"10.1021\/acs.analchem.1c02917","article-title":"Fast 19F magic-angle spinning nuclear magnetic\nresonance for the structural characterization of active pharmaceutical\ningredients in blockbuster drugs","volume":"93","author":"Quinn","year":"2021","journal-title":"Anal. Chem."},{"issue":"44","key":"2026091609105854900_cit16","doi-asserted-by":"publisher","first-page":"30483","DOI":"10.1021\/jacs.4c11373","article-title":"Integrating19F distance\nrestraints for accurate\nprotein structure determination by magic angle spinning NMR spectroscopy","volume":"146","author":"Runge","year":"2024","journal-title":"J. Am. Chem. Soc."},{"issue":"2","key":"2026091609105854900_cit17","doi-asserted-by":"publisher","first-page":"265","DOI":"10.1016\/0014-5793(75)80504-7","article-title":"NMR investigations of the dynamics of the aromatic\namino acid residues in the basic pancreatic trypsin inhibitor","volume":"50","author":"Wu\u0308thrich","year":"1975","journal-title":"FEBS Lett."},{"issue":"1","key":"2026091609105854900_cit18","doi-asserted-by":"publisher","first-page":"55","DOI":"10.1016\/0022-2860(95)08864-R","article-title":"Phenyl ring dynamics of the insulin\nfragment Gly-Phe-Phe(B23\u2013B25)\nby solid state deuterium NMR","volume":"355","author":"Naito","year":"1995","journal-title":"J. Mol. Struct."},{"issue":"18","key":"2026091609105854900_cit19","doi-asserted-by":"publisher","first-page":"3356","DOI":"10.1021\/jp983222g","article-title":"Phenyl ring\ndynamics of Enkephalin molecules and behavior of bound solvents in\nthe crystalline states by2H NMR spectroscopy","volume":"103","author":"Kamihira","year":"1999","journal-title":"J. Phys. Chem. A"},{"issue":"10","key":"2026091609105854900_cit20","doi-asserted-by":"publisher","DOI":"10.1002\/pro.70307","article-title":"19F NMR-based solvent\naccessibility profiling reveals tryptophan ring-flip dynamics in a\nprotein","volume":"34","author":"Kawagoe","year":"2025","journal-title":"Protein Sci."},{"issue":"3","key":"2026091609105854900_cit21","doi-asserted-by":"publisher","first-page":"388","DOI":"10.1021\/ja003220l","article-title":"Aromatic ring-flipping\nin supercooled water: implications for NMR-based structural biology\nof proteins","volume":"123","author":"Skalicky","year":"2001","journal-title":"J. Am. Chem. Soc."},{"issue":"19","key":"2026091609105854900_cit22","doi-asserted-by":"publisher","first-page":"4997","DOI":"10.1021\/acs.jpcb.7b02066","article-title":"Peptide\nand protein dynamics and\nlow-temperature\/DNP magic angle spinning NMR","volume":"121","author":"Ni","year":"2017","journal-title":"J. Phys. Chem. B"}],"container-title":["Journal of the American\nChemical Society"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jacs.6c10748","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/36\/38924\/68111471\/jacs.6c10748.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/36\/38924\/68111471\/jacs.6c10748.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T13:29:47Z","timestamp":1789565387000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jacsat\/article\/148\/36\/38924\/5396720\/Simultaneous-Site-and-Residue-Specific"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,1]]},"references-count":22,"journal-issue":{"issue":"36","published-online":{"date-parts":[[2026,9,1]]},"published-print":{"date-parts":[[2026,9,16]]}},"URL":"https:\/\/doi.org\/10.1021\/jacs.6c10748","relation":{},"ISSN":["0002-7863","1520-5126"],"issn-type":[{"value":"0002-7863","type":"print"},{"value":"1520-5126","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,1]]}}}