NMR protein structure determination in living E. Coli cells using nonlinear sampling

The cell is a crowded environment in which proteins interact specifically with other proteins, nucleic acids, cofactors and ligands. atomic resolution structural explanation of proteins functioning in this environment is a main goal of biochemical research.recent improvements to nuclear magnetic resonance (nMr) hardware and methodology allow the measurement of high-resolution heteronuclear multidimensional nMrspectra of macromolecules in living cells (in-cell nMr). In this study, we describe a protocol for the stable isotope (13c, 15nand 2H) labeling and structure determination of proteins overexpressed in Escherichia colicells exclusively on the basis of information obtained in living cells. the protocol combines the preparation of the protein in E. coli cells, the rapid measurement of the three-dimensional (3D) nMrspectra by nonlinear sampling of the indirectly acquired dimensions, structure calculation and structure refinement. under favorable circumstances, this in-cell nMrapproach can provide high-resolution 3D structures of proteins in living environments. the protocol has been used to solve the first 3D structure of a protein in living cells for the putative heavy metal-binding protein ttHa1718 from Thermus thermophilusHB8 overexpressed in E. colicells. as no protein purification is necessary, a sample for in-cell nMrmeasurements can be obtained within 2-3 d. With the nonlinear sampling scheme, the duration of each 3D experiment can be reduced to 2-3 h. once chemical shift assignments and noesY peak lists have been prepared, structure calculation with the program cYanaand energy refinement can be completed in less than 1 h on a powerful computer system.