Influence of Cell Type, Label Type, and Recovery Times on In-Cell Pulsed Dipolar Electron Paramagnetic Resonance Spectroscopy Data

In nature, most biomolecules perform their function in a cellular environment. It is therefore important to study their structure–dynamics–function relationships in cells and establish methods that can achieve this. Here, the suitability of pulsed dipolar electron paramagnetic resonance (EPR) spectroscopy (PDS) for such studies is tested in combination with different label and cell types. The model protein GB1 was labeled with either a trityl- or gadolinium-based spin label and transfected via electroporation into two types of mammalian cells, i.e., HeLa and HEK293, and one type of bacterial cells, i.e., E . coli . The in-cell Q-band PDS measurements revealed that both labels remain intact in all three cell types, whereas unspecific binding to the tested cell types was observed for the trityl-labeled protein when used in high concentrations, no such behavior was detectable for the gadolinium label due to its significantly lower sensitivity at Q-band. Interestingly, the modulation depth decreased for both labels with increasing cell recovery time, potentially indicating intracellular protein degradation or aggregation processes. In contrast, E . coli enabled substantially shorter recovery times and yielded PDS data with preserved modulation depth and distance information. Thus, in-cell PDS studies on spin-labeled proteins are particularly useful when combined with E . coli cells.