In-cell NMR spectroscopy in Escherichia coli

A living cell is a complex system that contains many biological macromolecules and small molecules necessary for survival, in a relatively small volume. It is within this crowded and complex cellular environment that proteins function making in-cell studies of protein structure and binding interactions an exciting and important area of study. Nuclear magnetic resonance (NMR) spectroscopy is a particularly attractive method for in-cell studies of proteins since it provides atomic-level data noninvasively in solution. In addition, NMR has recently undergone significant advances in instrumentation to increase sensitivity and in methods development to reduce data acquisition times for multidimensional experiments. Thus, NMR spectroscopy lends itself to studying proteins within a living cell, and recently "in-cell NMR" studies have been reported from several laboratories. To date, this technique has been successfully applied in Escherichia coli (E. coli), Xenopus laevis (X. laevis) oocytes, and HeLa host cells. Demonstrated applications include protein assignment as well as de novo 3D protein structure determination. The most common use, however, is to probe binding interactions and structural modifications directly from proton nitrogen correlation spectra. E. coli is the most extensively used cell type thus far and this chapter is largely confined to reviewing recent literature and describing methods and detailed protocols for in-cell NMR studies in this bacterial cell.