Abstract:
:EmrE is a small multidrug resistance transporter that has been well studied as a model for secondary active transport. Because transport requires the protein to convert between at least two states open to opposite sides of the membrane, it is expected that blocking these conformational transitions will prevent transport activity. We have previously shown that NMR can quantitatively measure the transition between the open-in and open-out states of EmrE in bicelles. Now, we have used the antiparallel EmrE crystal structure to design a cross-link to inhibit this conformational exchange process. We probed the structural, dynamic, and functional effects of this cross-link with NMR and in vivo efflux assays. Our NMR results show that our antiparallel cross-link performs as predicted: dramatically reducing conformational exchange while minimally perturbing the overall structure of EmrE and essentially trapping EmrE in a single state. The same cross-link also impairs ethidium efflux activity by EmrE in Escherichia coli. This confirms the hypothesis that transport can be inhibited simply by blocking conformational transitions in a properly folded transporter. The success of our cross-linker design also provides further evidence that the antiparallel crystal structure provides a good model for functional EmrE.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Dutta S,Morrison EA,Henzler-Wildman KAdoi
10.1016/j.bpj.2014.06.030subject
Has Abstractpub_date
2014-08-05 00:00:00pages
613-620issue
3eissn
0006-3495issn
1542-0086pii
S0006-3495(14)00673-0journal_volume
107pub_type
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