Observation of NMR Signals from Proteins Introduced into Living Mammalian Cells by Reversible Membrane Permeabilization Using a Pore-Forming Toxin,Streptolysin O

摘要

Structural information about proteins inside living cells is of crucial importance for understanding the biological events actually occurring within the intracellular environment, which are often difficult to reconstitute in an in vitro system. To achieve this goal, several "in-cell" NMR methods have been developed to directly detect the NMR signals derived from isotopically enriched molecules introduced within the living organisms. Using Escherichia coli cells overexpressing ~(13)C-and/or ~(15)N-labeled target proteins, investigations of protein-protein interactions, protein dynamics, and even the determination of the protein structure were performed within the "molecular crowding'' environment of the bacteria! cytosol, where the total protein concentration exceeds 200 mg/mL.