Tryptophan in the Pore of the Mechanosensitive Channel MscS

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 wi...

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Bibliographic Details
Published in:The Journal of biological chemistry 2010-02, Vol.285 (8), p.5377-5384
Main Authors: Rasmussen, Tim, Edwards, Michelle D., Black, Susan S., Rasmussen, Akiko, Miller, Samantha, Booth, Ian R.
Format: Article
Language:English
Subjects:
Biophysics
Escherichia Coli
Membrane/Channels
Methods/Fluorescence
Osmotic Stress
Phospholipid
Protein Conformation
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M109.071472