Specific ion effects on macromolecular interactions in Escherichia coli extracts

Ciara Kyne; Brian Ruhle; Virginie W. Gautier; Peter B. Crowley

doi:10.1002/pro.2615

Specific ion effects on macromolecular interactions in Escherichia coli extracts

Ciara Kyne, Brian Ruhle, Virginie W. Gautier, Peter B. Crowley

Cellular & Molecular Medicine

Research output: Contribution to a Journal (Peer & Non Peer) › Article › peer-review

36 Citations (Scopus)

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 Mg²⁺-containing buffers. Moreover, a combination of RNA digestion and Mg²⁺ greatly enhanced the NMR detection of ΔTat-GB1 in cell extracts.

Original language	English
Pages (from-to)	310-318
Number of pages	9
Journal	Protein Science
Volume	24
Issue number	3
DOIs	https://doi.org/10.1002/pro.2615
Publication status	Published - 1 Mar 2015

Keywords

arginine
cell extracts
in-cell NMR
protein interactions
size exclusion chromatography

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10.1002/pro.2615

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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 Mg2+-containing buffers. Moreover, a combination of RNA digestion and Mg2+ greatly enhanced the NMR detection of ΔTat-GB1 in cell extracts.",

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T1 - Specific ion effects on macromolecular interactions in Escherichia coli extracts

AU - Kyne, Ciara

AU - Ruhle, Brian

AU - Gautier, Virginie W.

AU - Crowley, Peter B.

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N2 - 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 Mg2+-containing buffers. Moreover, a combination of RNA digestion and Mg2+ greatly enhanced the NMR detection of ΔTat-GB1 in cell extracts.

AB - 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 Mg2+-containing buffers. Moreover, a combination of RNA digestion and Mg2+ greatly enhanced the NMR detection of ΔTat-GB1 in cell extracts.

KW - arginine

KW - cell extracts

KW - in-cell NMR

KW - protein interactions

KW - size exclusion chromatography

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Specific ion effects on macromolecular interactions in Escherichia coli extracts

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