Abstract
NhaA, the Na(+)/H(+) antiporter of Escherichia coli, exists in the native membrane as a homodimer of which two monomers have been suggested to be attached by a beta-hairpin at the periplasmic side of the membrane. Constructing a mutant deleted of the beta-hairpin, NhaA/Delta(Pro(45)-Asn(58)), revealed that in contrast to the dimeric mobility of native NhaA, the mutant has the mobility of a monomer in a blue native gel. Intermolecular cross-linking that monitors dimers showed that the mutant exists only as monomers in the native membrane, proteoliposomes, and when purified in beta-dodecyl maltoside micelles. Furthermore, pull-down experiments revealed that, whereas as expected for a dimer, hemagglutinin-tagged wild-type NhaA co-purified with His-tagged NhaA on a Ni(2+)-NTA affinity column, a similar version of the mutant did not. Remarkably, under routine stress conditions (0.1 m LiCl, pH 7 or 0.6 m NaCl, pH 8.3), the monomeric form of NhaA is fully functional. It conferred salt resistance to NhaA- and NhaB-deleted cells, and whether in isolated membrane vesicles or reconstituted into proteoliposomes exhibited Na(+)/H(+) antiporter activity and pH regulation very similar to wild-type dimers. Remarkably, under extreme stress conditions (0.1 m LiCl or 0.7 m NaCl at pH 8.5), the dimeric native NhaA was much more efficient than the monomeric mutant in conferring extreme stress resistance.
Highlights
NhaA is a 42-kDa integral membrane protein
Construction of the -Hairpin Deletion Mutant NhaA/ ⌬(Pro45-Asn58)—Constructing a monomeric NhaA can be most helpful in allowing a straightforward comparison between NhaA dimers and monomers with respect to functional and structural properties, and deduction thereof of the functional/ structural role of the dimeric state
The study revealed that the NhaA monomers are fully functional and stable in cells, isolated membranes, and reconstituted proteoliposomes
Summary
Bacterial Strains and Culture Conditions—EP432 is an E. coli K-12 derivative, which is melBLid, ⌬nhaA1::kan, ⌬nhaB1::cat, ⌬lacZY, thr1 [23]. Determination of the Accessibility to the Cross-linking Reagent—When no cross-linking was observed, it was crucial to ascertain that the Cys replacement was accessible to the reagent For this purpose, following cross-linking in the membranes, the beads with the affinity-purified protein were resuspended in 100 l of binding buffer (in the presence of the indicated DDM concentrations), containing 0.2 mM fluorescein 5-maleimide (Molecular Probes) and further incubated for 30 min at 25 °C to determine the free Cys [35]. When the accessibility of the purified NhaA variant was tested in the presence of the indicated concentrations of DDM, following cross-linking, 0.2 mM fluorescein 5-maleimide was added to the reaction mixture, and incubation continued for 30 min as above. All experiments were repeated at least twice with practically identical results
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