TY - JOUR
T1 - Site-directed tryptophan fluorescence reveals two essential conformational changes in the Na +/H + antiporter NhaA
AU - Kozachkov, Lena
AU - Padan, Etana
PY - 2011/10/20
Y1 - 2011/10/20
N2 - 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.
AB - 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.
KW - Membrane proteins
KW - Secondary transporters
KW - Transporter functional dynamics
UR - http://www.scopus.com/inward/record.url?scp=80053162595&partnerID=8YFLogxK
U2 - 10.1073/pnas.1109256108
DO - 10.1073/pnas.1109256108
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C2 - 21873214
AN - SCOPUS:80053162595
SN - 0027-8424
VL - 108
SP - 15769
EP - 15774
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 38
ER -