Figure 8. Membrane perturbations are largely eliminated upon MscS channel opening.

The figure summarizes the results from a 20-µs simulation of open MscS in a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane, using a coarse-grained representation. (A) The cryo-electron microscopy (EM) structure of MscS in the open state (blue cartoons) is overlaid with a calculated 3D density distribution mapping the morphology of the alkyl chain double layer in the molecular dynamics (MD) trajectory (gold volume), up to 50 Å from the protein surface. Protein and density maps are shown as in Figure 4. (B) Instantaneous configuration of the lipid bilayer in a snapshot of the MD trajectory, shown in cross-section as in Figure 5A. (C) Time-averages of the instantaneous lipid configurations observed in the trajectory, mapped across the membrane plane and shown in cross-section. Averages were calculated and are represented as in Figure 5B.

Figure 8—figure supplement 1. Change in protein-lipid interfacial area during gating is much smaller than what could be inferred from change of in-plane cross-sectional area.

(A) To evaluate the area of the protein surface exposed to the membrane, we quantified the number of coarse-grained (CG) particles in the channel within 6.5 Å of any lipid particle, for each snapshot of the molecular dynamics (MD) trajectories calculated for closed and open MscS in a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer. Note that each CG particle approximately represents one chemical group in protein sidechains and backbone and that a distance of 6.5 Å implies either a direct or close-range contact, as the typical radius of the CG particles is 2.6 Å. The results from this analysis are mapped on the channel surface, for either functional state, coloring each CG particle according to the persistence of their exposure to the lipid bilayer over time. (B) Histograms quantifying the variability in number of CG particles in the channel that are exposed to the lipid bilayer across different snapshots within the same trajectory. For completeness, data are shown for a distance threshold of 6.5 Å, as in panel (A), as well as for 5.2 Å, which implies direct contact. For threshold 5.2 Å, the number of lipid-contacting protein groups is 708.2±21.4, while in the open state, this number is 781.3±20.1, reflecting a 10.3% increase. For threshold 6.5 Å, the corresponding values are 1230.3±22.5 and 1267.9±17.6, reflecting a 3.0% difference. In contrast, geometric idealiziations of the protein-lipid interface would suggest a >90% increase in the in-plane cross-sectional area of the channel upon opening, and a >120% increase in the membrane-exposed area.

Figure 8—figure supplement 2. Changes in membrane morphology upon gating of MscS.

The figure summarizes results from simulations of alternative open conformations of wild-type and mutagenized MscS in POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Wang et al., 2008; Pliotas et al., 2015), as well as of a closed state, using a coarse-grained representation of the system (Table 1). The experimental structures (blue cartoons) are overlaid with calculated 3D density distributions mapping the morphology of the alkyl chain double layer in each of the molecular dynamics (MD) trajectories (gold volumes), up to 10 Å from the protein surface. Maps are represented as in Figure 4. Data represent averages over 20 µs of simulation.