Site-directed tryptophan fluorescence reveals two essential conformational changes in the Na +/H + antiporter NhaA

Lena Kozachkov, Etana Padan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

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 ligandinduced conformational change that does not occur until NhaA is activated at alkaline pH.

Original languageEnglish
Pages (from-to)15769-15774
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number38
DOIs
StatePublished - 20 Oct 2011

Keywords

  • Membrane proteins
  • Secondary transporters
  • Transporter functional dynamics

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