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Specific ion effects on macromolecular interactions inescherichia coliextracts

Kyne, Ciara
; 
Ruhle, Brian
; 
Gautier, Virginie W.
; 
Crowley, Peter B.
Repository DOI
Publication Date
2014-12-30
Keywords
arginine, cell extracts, in-cell nmr, protein interactions, size exclusion chromatography, cell nmr-spectroscopy, protein-protein interactions, nuclear-magnetic-resonance, in-cell, tat protein, living cells, hiv-1 tat, rna, recognition, stability
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Article
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Citation
Kyne, Ciara; Ruhle, Brian; Gautier, Virginie W. Crowley, Peter B. (2014). Specific ion effects on macromolecular interactions inescherichia coliextracts. Protein Science 24 (3), 310-318
Abstract
Protein characterization in situ remains a major challenge for protein science. Here, the interactions of Tat-GB1 in Escherichia coli cell extracts were investigated by NMR spectroscopy and size exclusion chromatography (SEC). Tat-GB1 was found to participate in high molecular weight complexes that remain intact at physiologically-relevant ionic strength. This observation helps to explain why Tat-GB1 was not detected by in-cell NMR spectroscopy. Extracts pre-treated with RNase A had a different SEC elution profile indicating that Tat-GB1 predominantly interacted with RNA. The roles of biological and laboratory ions in mediating macromolecular interactions were studied. Interestingly, the interactions of Tat-GB1 could be disrupted by biologically-relevant multivalent ions. The most effective shielding of interactions occurred in Mg2+-containing buffers. Moreover, a combination of RNA digestion and Mg2+ greatly enhanced the NMR detection of Tat-GB1 in cell extracts.
Funder
Publisher
Wiley-Blackwell
Publisher DOI
10.1002/pro.2615
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Attribution-NonCommercial-NoDerivs 3.0 Ireland
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Externally hosted open access publications with University of Galway authors (2)