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Protein Interactions in the Escherichia coli Cytosol: An Impediment to In‐Cell NMR Spectroscopy
Author(s) -
Crowley Peter B.,
Chow Elysian,
Papkovskaia Tatiana
Publication year - 2011
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100063
Subject(s) - nuclear magnetic resonance spectroscopy , escherichia coli , biochemistry , cytosol , chemistry , cytochrome c , cytochrome , mutant , biophysics , biology , stereochemistry , enzyme , mitochondrion , gene
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.