title: Site-directed tryptophan fluorescence reveals two essential conformational changes in the Na+/H+ antiporter NhaA.
abstract: NhaA, a Na(+)/H(+) antiporter critical for pH and Na(+) homeostasis in Escherichia coli, as well as other enterobacteria and possibly Homo sapiens, was modified for fluorescence spectroscopy by constructing a functional Trp-less NhaA mutant. Purified Trp-less NhaA lacks the Trp fluorescence emission characteristic of the wild type, thereby providing a background for studying structure-function relationships in NhaA by site-directed Trp fluorescence. Two single-Trp variants in the Trp-less background (F136W and F339W) were constructed. The mutants grow on selective media, have antiport activities that are similar to Trp-less NhaA, and exhibit Trp fluorescence with three different reversible responses to Li(+), Na(+), and/or pH. With single Trp/F136W, a pH shift from pH 6.0 to 8.5 induces a red shift and dramatically increases fluorescence in a reversible fashion; no effect is observed when either Na(+) or Li(+) is added. In marked contrast, with single Trp/F339W, changes in pH do not alter fluorescence, but addition of either Na(+) or Li(+) drastically quenches fluorescence at alkaline pH. Therefore, a Trp at position 136 specifically monitors a pH-induced conformational change that activates NhaA, whereas a Trp at position 339 senses a ligand-induced conformational change that does not occur until NhaA is activated at alkaline pH.
authors.affiliation: Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel.
authors.forename: Lena
authors.initials: L
authors.lastname: Kozachkov
authors.forename: Etana
authors.initials: E
authors.lastname: Padan
mesh_terms: D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008094:Lithium / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C102839:NhaA protein, E coli; D017923:Sodium-Hydrogen Exchangers; D014364:Tryptophan; D008094:Lithium; D012964:Sodium