Assessing oligomerization of membrane proteins by four-pulse DEER: pH-dependent dimerization of NhaA Na+/H+ antiporter of E. coli

Daniel Hilger, Heinrich Jung, Etana Padan, Christoph Wegener, Klaus Peter Vogel, Heinz Jürgen Steinhoff, Gunnar Jeschke*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

116 Scopus citations

Abstract

The pH dependence of the structure of the main Na+/H+ antiporter NhaA of Escherichia coli is studied by continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) techniques on singly spin-labeled mutants. Residues 225 and 254 were selected for site-directed spin labeling, as previous work suggested that they are situated in domains undergoing pH-dependent structural changes. A well-defined distance of 4.4 nm between residues H225R1 in neighboring molecules is detected by a modulation in double electron-electron resonance data. This indicates that NhaA exists as a dimer, as previously suggested by a low-resolution electron density map and cross-linking experiments. The modulation depth decreases reversibly when pH is decreased from 8 to 5.8. A quantitative analysis suggests a dimerization equilibrium, which depends moderately on pH. Furthermore, the mobility and polarity of the environment of a spin label attached to residue 225 change only slightly with changing pH, while no other changes are detected by CW EPR. As antiporter activity of NhaA changes drastically in the studied pH range, residues 225 and 254 are probably located not in the sensor or ion translocation sites themselves but in domains that convey the signal from the pH sensor to the translocation site.

Original languageEnglish
Pages (from-to)1328-1338
Number of pages11
JournalBiophysical Journal
Volume89
Issue number2
DOIs
StatePublished - Aug 2005

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