Detection of oligomerization and conformational changes in the Na +/H+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer

Akira Karasawa, Yumi Tsuboi, Hiroki Inoue, Rie Kinoshita, Norihiro Nakamura, Hiroshi Kanazawa

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Oligomerization and conformational changes in the Na+/H + antiporter from Helicobacter pylori (HPNhaA) were studied by means of fluorescence resonance energy transfer (FRET) analysis. Na+/H + antiporter-deficient Escherichia coli cells expressing C-terminal fusions of HPNhaA to green fluorescent protein (GFP) variants exhibited wild-type levels of antiporter activity in their everted membrane vesicles. Vesicles containing both HPNhaA-CFP and HPNhaA-YFP or HPNhaA-Venus exhibited FRET from CFP (donor) to YFP or Venus (acceptor), suggesting that HPNhaA forms an oligomer. Co-precipitation of HPNhaA tagged by Venus and FLAG sequences confirmed oligomerization. FRET decreased extensively after treatment of the vesicles with proteinase K, which released GFP variants from the fusion proteins. FRET was not observed by merely mixing vesicles expressing the donor or acceptor fusion alone. Fluorescence of Venus is less sensitive to anions and stronger than that of anion-sensitive YFP. Using HPNhaA-Venus as the acceptor, Li+ was found to cause a significant decrease in FRET regardless of the presence or absence of ΔpH across the membranes, whereas Na + caused a much weaker effect. This Li+ effect was minimal in vesicles prepared from cells expressing HPNhaA containing an Asp 141 to Asn mutation, which results in defective Li+/H + antiporter activity, possibly Li+ binding. These results demonstrate that monomer interactions within the HPNhaA oligomer are weakened possibly by Li+ binding. Dynamic interactions between HPNhaA monomers were detectable in membranes by FRET analysis, thus providing a new approach to study dynamic conformational changes in NhaA during antiport activity.

Original languageEnglish
Pages (from-to)41900-41911
Number of pages12
JournalJournal of Biological Chemistry
Volume280
Issue number51
DOIs
Publication statusPublished - Dec 23 2005
Externally publishedYes

Fingerprint

Venus
Oligomerization
Fluorescence Resonance Energy Transfer
Sodium-Hydrogen Antiporter
Helicobacter pylori
Antiporters
Fusion reactions
Green Fluorescent Proteins
Membranes
Oligomers
Anions
Monomers
Cells
Endopeptidase K
Ion Transport
Coprecipitation
Escherichia coli
Fluorescence
Mutation
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Detection of oligomerization and conformational changes in the Na +/H+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer. / Karasawa, Akira; Tsuboi, Yumi; Inoue, Hiroki; Kinoshita, Rie; Nakamura, Norihiro; Kanazawa, Hiroshi.

In: Journal of Biological Chemistry, Vol. 280, No. 51, 23.12.2005, p. 41900-41911.

Research output: Contribution to journalArticle

Karasawa, Akira ; Tsuboi, Yumi ; Inoue, Hiroki ; Kinoshita, Rie ; Nakamura, Norihiro ; Kanazawa, Hiroshi. / Detection of oligomerization and conformational changes in the Na +/H+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 51. pp. 41900-41911.
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abstract = "Oligomerization and conformational changes in the Na+/H + antiporter from Helicobacter pylori (HPNhaA) were studied by means of fluorescence resonance energy transfer (FRET) analysis. Na+/H + antiporter-deficient Escherichia coli cells expressing C-terminal fusions of HPNhaA to green fluorescent protein (GFP) variants exhibited wild-type levels of antiporter activity in their everted membrane vesicles. Vesicles containing both HPNhaA-CFP and HPNhaA-YFP or HPNhaA-Venus exhibited FRET from CFP (donor) to YFP or Venus (acceptor), suggesting that HPNhaA forms an oligomer. Co-precipitation of HPNhaA tagged by Venus and FLAG sequences confirmed oligomerization. FRET decreased extensively after treatment of the vesicles with proteinase K, which released GFP variants from the fusion proteins. FRET was not observed by merely mixing vesicles expressing the donor or acceptor fusion alone. Fluorescence of Venus is less sensitive to anions and stronger than that of anion-sensitive YFP. Using HPNhaA-Venus as the acceptor, Li+ was found to cause a significant decrease in FRET regardless of the presence or absence of ΔpH across the membranes, whereas Na + caused a much weaker effect. This Li+ effect was minimal in vesicles prepared from cells expressing HPNhaA containing an Asp 141 to Asn mutation, which results in defective Li+/H + antiporter activity, possibly Li+ binding. These results demonstrate that monomer interactions within the HPNhaA oligomer are weakened possibly by Li+ binding. Dynamic interactions between HPNhaA monomers were detectable in membranes by FRET analysis, thus providing a new approach to study dynamic conformational changes in NhaA during antiport activity.",
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T1 - Detection of oligomerization and conformational changes in the Na +/H+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer

AU - Karasawa, Akira

AU - Tsuboi, Yumi

AU - Inoue, Hiroki

AU - Kinoshita, Rie

AU - Nakamura, Norihiro

AU - Kanazawa, Hiroshi

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N2 - Oligomerization and conformational changes in the Na+/H + antiporter from Helicobacter pylori (HPNhaA) were studied by means of fluorescence resonance energy transfer (FRET) analysis. Na+/H + antiporter-deficient Escherichia coli cells expressing C-terminal fusions of HPNhaA to green fluorescent protein (GFP) variants exhibited wild-type levels of antiporter activity in their everted membrane vesicles. Vesicles containing both HPNhaA-CFP and HPNhaA-YFP or HPNhaA-Venus exhibited FRET from CFP (donor) to YFP or Venus (acceptor), suggesting that HPNhaA forms an oligomer. Co-precipitation of HPNhaA tagged by Venus and FLAG sequences confirmed oligomerization. FRET decreased extensively after treatment of the vesicles with proteinase K, which released GFP variants from the fusion proteins. FRET was not observed by merely mixing vesicles expressing the donor or acceptor fusion alone. Fluorescence of Venus is less sensitive to anions and stronger than that of anion-sensitive YFP. Using HPNhaA-Venus as the acceptor, Li+ was found to cause a significant decrease in FRET regardless of the presence or absence of ΔpH across the membranes, whereas Na + caused a much weaker effect. This Li+ effect was minimal in vesicles prepared from cells expressing HPNhaA containing an Asp 141 to Asn mutation, which results in defective Li+/H + antiporter activity, possibly Li+ binding. These results demonstrate that monomer interactions within the HPNhaA oligomer are weakened possibly by Li+ binding. Dynamic interactions between HPNhaA monomers were detectable in membranes by FRET analysis, thus providing a new approach to study dynamic conformational changes in NhaA during antiport activity.

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