Peer-Reviewed Journal Details
Mandatory Fields
Crowley, PB,Chow, E,Papkovskaia, T
2011
May
Chembiochem
Protein Interactions in the Escherichia coli Cytosol: An Impediment to In-Cell NMR Spectroscopy
Published
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Optional Fields
chemical biology chromatography electrostatic interactions NMR spectroscopy protein-protein interactions NUCLEAR-MAGNETIC-RESONANCE CYTOCHROME-C LIVING CELLS CRYSTAL-STRUCTURE BINDING-SITE COMPLEXES ISO-1-CYTOCHROME-C DISSOCIATION PLASTOCYANIN EXPRESSION
12
1043
1048
Protein science is shifting towards experiments performed under native or native-like conditions. In-cell NMR spectroscopy for instance has the potential to reveal protein structure and dynamics inside cells. However, not all proteins can be studied by this technique. (15)N-labelled cytochrome c (cyt c) over-expressed in Escherichia coli was undetectable by in-cell NMR spectroscopy. When whole-cell lysates were subjected to size-exclusion chromatography (SEC) cyt c was found to elute with an apparent molecular weight of > 150 kDa. The presence of high molecular weight species is indicative of complex formation between cyt c and E. coli cytosolic proteins. These interactions were disrupted by charge-inverted mutants in cyt c and by elevated concentrations of NaCl. The physiologically relevant salt, KGlu, was less efficient at disrupting complex formation. Notably, a triple mutant of cyt c could be detected in cell lysates by NMR spectroscopy. The protein, GB1, yields high quality in-cell spectra and SEC analysis of lysates containing GB1 revealed a lack of interaction between GB1 and E. coli proteins. Together these data suggest that protein "stickiness" is a limiting factor in the application of in-cell NMR spectroscopy.
10.1002/cbic.201100063
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