Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis

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.

Figure 1

Stability of MscS tryptophan mutants. (A) Western blot of E. coli MscS Trp mutants in which 15 μg of membrane protein was loaded on a 4 to 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and after development Western blotting with antibody specific for the His₆ tag (see Experimental Procedures). (B) Membrane proteins (30 μg) were solubilized in PBS (pH 7.4) containing 1% DDM, 2.8 M urea, and 1 mM EDTA and prepared for BN-PAGE as described in Experimental Procedures. Samples were separated on Novex 4 to 16% Bis-Tris gradient native gels (Invitrogen) and proteins detected by Western blot as described for panel A. The positions of heptameric and monomeric MscS with associated lipids and detergent are indicated.

Figure 2

Size exclusion chromatographs of selected MscS tryptophan mutants. Heptameric and monomeric MscS were separated on a HiPrep Superdex 200 10/600 GL size exclusion column as described in Experimental Procedures. A vertical dashed line indicates the heptamer position for WT MscS.

Figure 3

Protection against hypoosmotic shock. Transformants of MJF612 (lacking MscL, MscS, MscK, and YbdG) expressing individual MscS Trp mutants were assessed for survival after a 0.5 M NaCl hypoosmotic shock. Mutants were either expressed as a result of the escape promoter activity of the pTrc promoter (A) or induced during growth (B; 0.3 mM IPTG for 30 min) immediately prior to downshock (see Experimental Procedures). Data are means and the standard deviation of three independent cultures.

Figure 4

Representative electrophysiology traces for selected Trp mutants. When assayed by patch clamp electrophysiology on isolated patches, Trp mutants were found to have four different types of behavior. Three are shown here: (a) WT-like, (b) short dwell, and (c) variable conductance (the fourth type of behavior was the lack of channel activity in the patch other than MscL, and these data are not shown). Additional data are shown in Figure S1 of the Supporting Information.

Figure 5

V99W generates two distinct open states. (A) Opening of V99W at “normal pressures” showed short dwell openings with WT-like conductance (one asterisk, ∼24 pA), but as the pressure was increased, channel openings (two asterisks) were associated with higher conductance and stable openings were observed (∼34 pA, ∼1.7 nS) (top). In the double mutant, V99W/F80A, only stable conductances of WT magnitude (∼23 pA, ∼1.2 nS) were observed (bottom). The high conductance may be explained by a π–π interaction between V99W and F80 on adjacent subunits (i.e., V99W:a with F80A:b) in the open state (B; PDB entry 2VV5), whereas the residues are well-separated in the closed state (C; PDB entry 2OAU) (V99W is colored red and F80 green). Only two subunits are shown for the sake of clarity. Models and figures were produced with PyMol. (D) Current-voltage plot for V99W at low (*; see panel 5A) (Black open squares) and high (**; see panel 5A) (red open circles) pressure, as described in text.

Figure 6

Relationship between the λ_max for Trp fluorescence and K_SV, the Stern–Volmer constant. Fluorescence spectroscopy was performed as described previously^, on DDM-solubilized proteins. For λ_max measurement, Trp residues were excited at 295 nm with a 2 nm slit width and emission was recorded between 300 and 420 nm with a slit width of 2 nm. A 0.2 mL microcuvette was used with 10 mm excitation and 4 mm emission path lengths. For K_SV, quenching by acrylamide in the concentration range of 0–0.2 M was quantified (see Experimental Procedures). The data for L105W and L109W are colored red.

Figure 7

Water accessibility and fluorescence anisotropy of the MscS pore surface. (A) Accessibility to the bulk water phase was detected by quenching of the tryptophan fluorescence by acrylamide. Strong quenching is colored red and weak quenching blue. (B) Steady-state fluorescence anisotropy is shown where blue indicates low values and red high values. If the mobility is comparable for all mutants, the pore shape is reflected by anisotropy because of the changing efficiency of homo-Förster resonance energy transfer. For the sake of clarity, only five of the seven subunits are shown. The figures were generated with PyMol on the basis of the closed crystal structure (PDB entry 2OAU).

Figure 8

Emission maxima of selected MscS mutants in DDM and reconstituted into membrane bilayers. Six Trp mutants were purified and reconstituted into DOPC bilayers and the emission spectra recorded as described in Experimental Procedures. (A) The red-shift of the emission maxima can be seen in particular for residues in the hydrophobic section of the pore. (B) The positions of the residues are shown on the closed structure 2OAU. (C) Emission spectra of DDM-solubilized samples (black) and samples reconstituted into DOPC (red) are compared.