Studying Proteins Inside Eukaryotic Cells in NMR

Abstract

In-cell NMR spectroscopy is a method used to observe isotopically labeled molecules within living cells. The first in-cell NMR experiment was performed with an E. coli overexpressing a 15N-labeled protein. For the first application of the in-cell NMR method with eukaryotic cells, isotopically labeled target proteins were introduced, by microinjection, into Xenopus laevis oocytes, and a cell-penetrating tag was also utilized. Our group reported an in-cell NMR method for mammalian cells; we used a pore-forming toxin, streptolysin O (SLO), to introduce target proteins by diffusion. By using these methods, protein-drug interactions and intracellular post-translational modifications, such as phosphorylation and acetylation, were successfully detected in vivo. However, the major limitation of the in-cell NMR experiments is the occurrence of cell death during the NMR measurement. As the suspension contains a high density of cells, nutrient depletion occurs rapidly in the anaerobic environment within the NMR tube, thus causing the deterioration of conditions and resulting in cell death during NMR measurements. Therefore, in-cell NMR experiments for eukaryotic cells currently have limited applications, such as for obtaining a single NMR spectrum measured within a very short time. Although sparse sampling methods have been utilized to shorten the time required to acquire multidimensional NMR spectra, many existing in vitro NMR experiments that are used to provide information regarding dynamics and protein interactions take several hours to perform. In this study, we will show a bioreactor system for in-cell NMR, which we developed, to suppress the cell death during NMR measurements, and its recent application in our lab.