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However, commonly used 19F-containing UAAs often suffer from narrow chemical shift dispersion and low sensitivity to environmental or protein conformational changes, especially in complex cellular conditions. Herein, we report a 19F-labeled unnatural amino acid, monofluoromethylphenylalanine (mfmF, CH2F-Phe), which displays superior spectroscopic properties for both in vitro and in-cell 19F NMR studies. Using genetic code expansion, CH2F-Phe was efficiently site-specifically incorporated into two model proteins light-oxygen-voltage sensing domain 2 (LOV2) and Calmodulin (CaM). Compared to trifluoromethylphenylalanine (CF3-Phe), CH2F-Phe exhibits broader 19F chemical shift dispersion and markedly enhanced sensitivity to environmental changes. Using this synthesized 19F NMR probe, we successfully achieved sensitive detection of the light-induced conformational transition of LOV2 in vitro. Furthermore, CH2F-Phe-incorporated CaM displayed remarkably sensitive and substantially larger 19F chemical shift differences (&amp;gt;1.0\u20132.0 ppm) between the apo- and Ca2+-bound states compared to CF3-Phe-labeled CaM, enabling the detection of previously unresolved intermediate states during Ca2+-dependent conformational transitions in living cells. Collectively, CH2F-Phe serves as an invaluable probe to characterize protein dynamics and interactions.<\/jats:p>","DOI":"10.1021\/jacs.6c12448","type":"journal-article","created":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T03:01:35Z","timestamp":1790910095000},"page":"43356-43365","source":"Crossref","is-referenced-by-count":0,"title":["Monofluoromethylphenylalanine:\nA Sensitive 19F Probe for Protein Conformational Dynamics\n                    <i>In Vitro<\/i>\n                    and in Cells"],"prefix":"10.1021","volume":"148","author":[{"given":"Pan","family":"Shi","sequence":"first","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, P. R. China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jun","family":"Liang","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, P. R. China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6003-9455","authenticated-orcid":true,"given":"XiaoLi","family":"Liu","sequence":"additional","affiliation":[{"name":"Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , ,","place":["Wuhan, P. R. China, 430071"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Zhang","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, P. R. China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0024-1096","authenticated-orcid":true,"given":"Chaowei","family":"Shi","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, P. R. China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sen","family":"Guo","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, P. R. China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiacai","family":"Song","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, P. R. China, 230026"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9315-900X","authenticated-orcid":true,"given":"Changlin","family":"Tian","sequence":"additional","affiliation":[{"name":"University of Science and Technology of China , , ,","place":["Hefei, P. R. China, 230026"]},{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, P. R.China, 230024"]},{"name":"Beijing Life Science Academy , ,","place":["Beijing, P. R. China, 102209"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5798-1722","authenticated-orcid":true,"given":"Conggang","family":"Li","sequence":"additional","affiliation":[{"name":"Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences , , ,","place":["Wuhan, P. R. 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