Structure and Dynamics of a Protein Inside Endosomal Vesicles in Intact Human Cells

With in-cell NMR, the structural and dynamic features of proteins can be analyzed directly in living cells at atomic resolution. However, in mammalian cells, such as human cells, the structural/dynamic features and physiological functions are not fully understood at atomic resolution. In this study, we developed a method for the highly viable and efficient introduction of stable isotope-labeled proteins into human cells by electrotransfection. Thus, it was possible to perform in-cell NMR to study the structural/dynamic features of intracellular proteins with a time resolution of a minute. The transfected proteins were localized in intracellular vesicles and gradually degraded as the vesicles acidified, which could be controlled by adding EDTA, a membrane-impermeable low molecular weight compound, during electrotransfection. With this method, we could then determine the precise structure and dynamics of proteins in endosomal vesicles. Our approach demonstrates that the combination of electrotransfection and in-cell NMR provides an opportunity to study the structural/dynamic features and physiological functions of molecules taken into intact cells.