title: Specific ion effects on macromolecular interactions in Escherichia coli extracts.
abstract: Protein characterization in situ remains a major challenge for protein science. Here, the interactions of ΔTat-GB1 in Escherichia coli cell extracts were investigated by NMR spectroscopy and size exclusion chromatography (SEC). ΔTat-GB1 was found to participate in high molecular weight complexes that remain intact at physiologically-relevant ionic strength. This observation helps to explain why ΔTat-GB1 was not detected by in-cell NMR spectroscopy. Extracts pre-treated with RNase A had a different SEC elution profile indicating that ΔTat-GB1 predominantly interacted with RNA. The roles of biological and laboratory ions in mediating macromolecular interactions were studied. Interestingly, the interactions of ΔTat-GB1 could be disrupted by biologically-relevant multivalent ions. The most effective shielding of interactions occurred in Mg(2+) -containing buffers. Moreover, a combination of RNA digestion and Mg(2+) greatly enhanced the NMR detection of ΔTat-GB1 in cell extracts.
authors.affiliation: School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland.
authors.forename: Ciara
authors.initials: C
authors.lastname: Kyne
authors.forename: Brian
authors.initials: B
authors.lastname: Ruhle
authors.forename: Virginie W
authors.initials: VW
authors.lastname: Gautier
authors.forename: Peter B
authors.initials: PB
authors.lastname: Crowley
mesh_terms: D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D002457:Cell Extracts / Q000737:chemistry; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry*; D019913:Nuclear Localization Signals / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D009994:Osmolar Concentration; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D002457:Cell Extracts; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D001120:Arginine; D008274:Magnesium
keywords: arginine; cell extracts; in-cell NMR; protein interactions; size exclusion chromatography