Figure 4: Probing the functional role of the N-terminal domain and the physical interaction between the N terminus and TM2 by deletion analysis.

(a) Survival of MJF465 (MscL⁻ MscS⁻ MscK⁻) E. coli expressing different deletion constructs of MscL after downshock from LB supplemented with 500 mM NaCl to LB (∼1,000 mOsm). Data shown as box plots indicating the mean, 25 and 75 percentile (box) and ±s.d. (capped lines). (b) Midpoint of pressure activation of individual deletion constructs determined from multichannel patches (n=4, Δ2–7, n=3; mean±s.d.). The dotted horizontal line represents the mean value for WT MscL. (c) Local dynamics at the intracellular end of TM2 (spin-labelled at position M94) as a function of N-terminal deletions. Left, cartoon representation of subunits i and i+2 showing the position of the spin-labelling site (blue sphere) and the individual residues in the N terminus that were deleted (red spheres). Right, EPR spectra of M94-SL in a WT background, four different deletions (Δ2–4, Δ2–5, Δ2–6 and Δ2–7) and the WT background opened in the presence of LPCs. The N-terminal sequence of MscL is shown with the region of deleted residues in red. Bar represents 20 G. (d) Calculated mobility parameter at position M94-SL for the different spin-labelled constructs in a,c. (e) A cartoon representation of the electrostatic interaction of the Glu (E6 and E9) residues on subunits i with Lys (K97) residue of the second adjacent (i+2) subunit. (f) A cartoon representation of the electrostatic interaction of the Lys (K5) residue on subunits i with Glu (E108) residue of the adjacent (i+1) subunit and phosphate group of a POPE lipid molecule. The position of M94 has been indicated as a purple sphere with respect to these residues in e,f.