Journal of Biological Chemistry
Volume 285, Issue 8, 19 February 2010, Pages 5377-5384
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Membrane Biology
Tryptophan in the Pore of the Mechanosensitive Channel MscS: ASSESSMENT OF PORE CONFORMATIONS BY FLUORESCENCE SPECTROSCOPY*

https://doi.org/10.1074/jbc.M109.071472Get rights and content
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Structural changes in channel proteins give critical insights required for understanding the gating transitions that underpin function. Tryptophan (Trp) is uniquely sensitive to its environment and can be used as a reporter of conformational changes. Here, we have used site-directed Trp insertion within the pore helices of the small mechanosensitive channel protein, MscS, to monitor conformational transitions. We show that Trp can be inserted in place of Leu at the two pore seal positions, Leu105 and Leu109, resulting in functional channels. Using Trp105 as a probe, we demonstrate that the A106V mutation causes a modified conformation in the purified channel protein consistent with a more open state in solution. Moreover, we show that solubilized MscS changes to a more open conformation in the presence of phospholipids or their lysoforms.

Biophysics
Membrane/Channels
Methods/Fluorescence
Phospholipid
Protein Conformation
Escherichia Coli
Osmotic Stress

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*

This work was supported by The Wellcome Trust (Grant 040174 and 086903).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.