Assessing Oligomerization of Membrane Proteins by Four-Pulse DEER: pH-dependent Dimerization of NhaA Na+/H+ antiporter of E. coli

¹ LMU München, Department Biologie I, D-80638 München, Germany
² Hebrew University of Jerusalem, Alexander Silberman Institute of Life Sciences, Jerusalem, Israel
³ Universität Osnabrück, Fachbereich Physik, D-49069 Osnabrück, Germany
⁴ Universitaet Osnabrueck
⁵ Max-Planck-Institute for Polymer research

^* To whom correspondence should be addressed. E-mail: jeschke{at}mpip-mainz.mpg.de.

Submitted on March 2, 2005
Revised on April 15, 2005
Accepted on 11 May 2005

	Abstract

The pH dependence of the structure of the main Na+/H+ antiporter NhaA of E. 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 (DEER) 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.

Key Words: EPR, dimerization equilibrium, protein aggregation, regulation, secondary transporter, site-directed spin labeling

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