Monofluoromethylphenylalanine: A Sensitive 19F Probe for Protein Conformational Dynamics In Vitro and in Cells.

The combination of site-specific incorporation of 19F-labeled unnatural amino acids (UAAs) and 19F nuclear magnetic resonance (NMR) spectroscopy is a powerful strategy for investigating protein structure, dynamics, and interactions. 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 (>1.0-2.0 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.