title: Tryptophan in the pore of the mechanosensitive channel MscS: assessment of pore conformations by fluorescence spectroscopy.
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.
authors.affiliation: School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, United Kingdom. t.rasmussen@abdn.ac.uk
authors.forename: Tim
authors.initials: T
authors.lastname: Rasmussen
authors.forename: Michelle D
authors.initials: MD
authors.lastname: Edwards
authors.forename: Susan S
authors.initials: SS
authors.lastname: Black
authors.forename: Akiko
authors.initials: A
authors.lastname: Rasmussen
authors.forename: Samantha
authors.initials: S
authors.lastname: Miller
authors.forename: Ian R
authors.initials: IR
authors.lastname: Booth
mesh_terms: D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary / Q000502:physiology; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; C469542:MscS protein, E coli; D010743:Phospholipids; D014364:Tryptophan