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Specific ion effects on macromolecular interactions in E scherichia coli extracts
Author(s) -
Kyne Ciara,
Ruhle Brian,
Gautier Virginie W.,
Crowley Peter B.
Publication year - 2015
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2615
Subject(s) - rnase p , chemistry , macromolecule , nuclear magnetic resonance spectroscopy , rna , escherichia coli , biophysics , size exclusion chromatography , biochemistry , biology , stereochemistry , enzyme , gene
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.