An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca²⁺ concentration in HeLa cells

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca²⁺-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca²⁺ concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca²⁺ concentration during experiments, human calbindin D_9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells.Time-resolved measurements of 2D ¹H-¹⁵N SOFAST-HMQC experiments of calbindin D_9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D_9k (P47M+C80) is initially in the Mg²⁺-bound state, and then gradually converted to the Ca²⁺-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca²⁺ into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies.