Tryptophan in the pore of the mechanosensitive channel MscS

assessment of pore conformations by fluorescence spectroscopy

Tim Rasmussen, Michelle Diane Edwards, Susan Shirley Black, Akiko Rasmussen, Samantha Miller, Ian Rylance Booth

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

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, Leu(105) and Leu(109), resulting in functional channels. Using Trp(105) 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.

Original languageEnglish
Pages (from-to)5377-5384
Number of pages8
JournalThe Journal of Biological Chemistry
Volume285
Issue number8
DOIs
Publication statusPublished - 19 Feb 2010

Keywords

  • ELECTRONIC-ENERGY TRANSFER
  • ESCHERICHIA-COLI
  • POLARIZATION SPECTRUM
  • CYCLIC HEXAPEPTIDES
  • MEMBRANE-PROTEIN
  • ION CHANNELS
  • RESOLUTION
  • TRANSITIONS
  • MECHANISM
  • RESIDUES

Cite this

Tryptophan in the pore of the mechanosensitive channel MscS : assessment of pore conformations by fluorescence spectroscopy. / Rasmussen, Tim; Edwards, Michelle Diane; Black, Susan Shirley; Rasmussen, Akiko; Miller, Samantha; Booth, Ian Rylance.

In: The Journal of Biological Chemistry, Vol. 285, No. 8, 19.02.2010, p. 5377-5384.

Research output: Contribution to journalArticle

Rasmussen, Tim ; Edwards, Michelle Diane ; Black, Susan Shirley ; Rasmussen, Akiko ; Miller, Samantha ; Booth, Ian Rylance. / Tryptophan in the pore of the mechanosensitive channel MscS : assessment of pore conformations by fluorescence spectroscopy. In: The Journal of Biological Chemistry. 2010 ; Vol. 285, No. 8. pp. 5377-5384.
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AU - Miller, Samantha

AU - Booth, Ian Rylance

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KW - ELECTRONIC-ENERGY TRANSFER

KW - ESCHERICHIA-COLI

KW - POLARIZATION SPECTRUM

KW - CYCLIC HEXAPEPTIDES

KW - MEMBRANE-PROTEIN

KW - ION CHANNELS

KW - RESOLUTION

KW - TRANSITIONS

KW - MECHANISM

KW - RESIDUES

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