Evaluation of parameters critical to observing proteins inside living Escherichia coli by in-cell NMR spectroscopy.
Our recently developed in-cell NMR procedure now enables one to observe protein conformations inside living cells. Optimization of the technique demonstrates that distinguishing the signals produced by a single protein species depends critically on protein overexpression levels and the correlation time in the cytoplasm. Less relevant is the selective incorporation of (15)N. Poorly expressed proteins, insoluble proteins, and proteins that cannot tumble freely due to associations within the cell cannot yet be observed. We show in-cell NMR spectra of bacterial NmerA and human calmodulin and discuss limitations of the technique as well as prospects for future applications.
Serber
Z
Z
Graduate Group in Biophysics, Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94143, USA.
Ledwidge
R
R
Miller
S M
SM
Dötsch
V
V
D000596:Amino Acids / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D012293:Rifampin / Q000494:pharmacology
D000596:Amino Acids; D001426:Bacterial Proteins; D002147:Calmodulin; D004791:Enzyme Inhibitors; D009587:Nitrogen Isotopes; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin