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 NMR spectra of macromolecules in living cells (in-cell NMR). In this study, we describe a protocol for the stable isotope ((13)C, (15)N and (2)H) labeling and structure determination of proteins overexpressed in Escherichia coli cells 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) NMR spectra by nonlinear sampling of the indirectly acquired dimensions, structure calculation and structure refinement. Under favorable circumstances, this in-cell NMR approach 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 thermophilus HB8 overexpressed in E. coli cells. As no protein purification is necessary, a sample for in-cell NMR measurements 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 CYANA and energy refinement can be completed in less than 1 h on a powerful computer system.
Department of Chemistry, Tokyo Metropolitan University, Hachioji, Tokyo, Japan.
Teppei
T
Ikeya
Atsuko
A
Sasaki
Daisuke
D
Sakakibara
Yoshiki
Y
Shigemitsu
Junpei
J
Hamatsu
Tomomi
T
Hanashima
Masaki
M
Mishima
Masatoshi
M
Yoshimasu
Nobuhiro
N
Hayashi
Tsutomu
T
Mikawa
Daniel
D
Nietlispach
Markus
M
Wälchli
Brian O
BO
Smith
Masahiro
M
Shirakawa
Peter
P
Güntert
Yutaka
Y
Ito
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002247:Carbon Isotopes; D003198:Computer Simulation; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012984:Software; D016963:Thermus thermophilus / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D011994:Recombinant Proteins