Abstract
Bacterial 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.