Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy.
The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.g., bacterial cells grown on isotopically labeled media), or by injecting isotopically labeled proteins directly into either cells or cell extracts. Here we provide detailed protocols for in-cell NMR experiments in the prokaryotic organism Escherichia coli, as well as eukaryotic cells and extracts employing Xenopus laevis oocytes or egg extracts. In-cell NMR samples with proteins overexpressed in E. coli can be produced within 13-14 h. Preparing Xenopus oocyte samples for in-cell NMR experiments takes 6-14 h depending on the oocyte preparation scheme and the injection method used.
Serber
Zach
Z
Department of Chemical and Systems Biology, Stanford University Medical School, 269 Campus Drive, Stanford, 94305 California, USA.
Selenko
Philipp
P
Hänsel
Robert
R
Reckel
Sina
S
Löhr
Frank
F
Ferrell
James E
JE
Wagner
Gerhard
G
Dötsch
Volker
V
D000818:Animals; D002478:Cells, Cultured; D004926:Escherichia coli / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D046911:Macromolecular Substances / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010063:Ovum / Q000737:chemistry; D014982:Xenopus laevis
D046911:Macromolecular Substances