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Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis
journal contribution
posted on 2015-07-28, 00:00 authored by Tim Rasmussen, Akiko Rasmussen, Shivani Singh, Heloisa Galbiati, Michelle
D. Edwards, Samantha Miller, Ian R. BoothBacterial mechanosensitive channels
gate when the transmembrane
turgor rises to levels that compromise the structural integrity of
the cell wall. Gating creates a transient large diameter pore that
allows hydrated solutes to pass from the cytoplasm at rates close
to those of diffusion. In the closed conformation, the channel limits
transmembrane solute movement, even that of protons. In the MscS crystal
structure (Protein Data Bank entry 2oau), a narrow, hydrophobic opening is visible
in the crystal structure, and it has been proposed that a vapor lock
created by the hydrophobic seals, L105 and L109, is the barrier to
water and ions. Tryptophan scanning mutagenesis has proven to be a
highly valuable tool for the analysis of channel structure. Here Trp
residues were introduced along the pore-forming TM3a helix and in
selected other parts of the protein. Mutants were investigated for
their expression, stability, and activity and as fluorescent probes
of the physical properties along the length of the pore. Most Trp
mutants were expressed at levels similar to that of the parent (MscS
YFF) and were stable as heptamers in detergent in the presence and
absence of urea. Fluorescence data suggest a long hydrophobic region
with low accessibility to aqueous solvents, extending from L105/L109
to G90. Steady-state fluorescence anisotropy data are consistent with
significant homo-Förster resonance energy transfer between
tryptophan residues from different subunits within the narrow pore.
The data provide new insights into MscS structure and gating.
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Tryptophan scanning mutagenesischannel structureTMtransmembrane turgorFluorescence dataTrp mutantsdiameter poretryptophan residueschannel limits transmembrane solute movementMscS crystal structureTrp residuesMscS YFFL 105Mechanosensitive Channel MscS Pore Revealedcrystal structureMscS structurecell wallTryptophan Scanning MutagenesisBacterial mechanosensitive channels gate
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