Detection of oligomerization and conformational changes in the Na+/H+ antiporter from Helicobacter pylori by fluorescence resonance energy transfer.
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 DeltapH across the membranes, whereas Na+ caused a much weaker effect. This Li+ effect was minimal in vesicles prepared from cells expressing HPNhaA containing an Asp141 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.
Karasawa
Akira
A
Department of Biological Sciences, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Tsuboi
Yumi
Y
Inoue
Hiroki
H
Kinoshita
Rie
R
Nakamura
Norihiro
N
Kanazawa
Hiroshi
H
D001483:Base Sequence; D001704:Biopolymers / Q000737:chemistry*; D011232:Chemical Precipitation; D017931:DNA Primers; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry; D016480:Helicobacter pylori / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000378:metabolism; D011487:Protein Conformation; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000378:metabolism
D001704:Biopolymers; D017931:DNA Primers; D017923:Sodium-Hydrogen Exchangers; D049452:Green Fluorescent Proteins; D008094:Lithium