title: Protein interactions in the Escherichia coli cytosol: an impediment to in-cell NMR spectroscopy.
abstract: 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.
authors: School of Chemistry, NUI Galway, Ireland. peter.crowley@nuigalway.ie
authors: Peter B
authors: PB
authors: Crowley
authors: Elysian
authors: E
authors: Chow
authors: Tatiana
authors: T
authors: Papkovskaia
mesh_terms: D002850:Chromatography, Gel; D045304:Cytochromes c / Q000096:biosynthesis / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008970:Molecular Weight; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding
chemical_list: D029968:Escherichia coli Proteins; D045304:Cytochromes c