In-cell NMR spectroscopy is a powerful tool to investigate protein behavior in physiologically relevant environments. In contrast to 2D 1 H- 15 N HSQC spectra of globular proteins that are plagued by substantial line broadening in mammalian cells, 1D 19 F spectra are readily observed. This presents an opportunity to assess intracellular pH in the cellular environment by 19 F NMR. For example, measuring variations in intracellular pH can provide invaluable insights into cellular and metabolic health since metabolic processes are heavily influenced by intracellular and organelle-level pH values. We are developing an effective strategy to measure intracellular pH changes over the time course of in-cell NMR measurements. Using several proteins, we demonstrate that fluorinated tryptophan (F-trp) residues can serve as exquisite probes for pH measurement. Using both globular (drkN-SH3 domain) and disordered (α-synuclein) model proteins, we established that F-trp residues strategically placed on surface accessible sites can report on pH changes in the physiologically relevant range of 6.0–7.7 for different cell types. Finally, we devised a short, six-residue flexible amino acid tag containing a fluorinated Trp that, when added to proteins provide an effective means to measure intracellular pH differences. In this manner, we can test for changesfor changes in the cellular milieu.
Lavrenyuk et al. (Sun,) studied this question.