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To characterize mechanosensitive channels functionally we patch-clamp bacteria.  For this purpose we generate giant spheroplasts (1). MscL single-channel currents (2) are activated by suction, they are large (70 pS at 20 mV) and display subconducting states. MscL survives solubilization, purification and reconstitution into liposomes. Panel 3 shows the activation curve recorded at different membrane tensions from a multi-channel liposome patch (from Sukharev, Sigurdson et al., 1999).

       

 

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Models of the E. coli MscL in the closed, expanded and open states created in collaboration with Dr. H.R. Guy. The model of the closed conformation was generated by homology after the crystal structure of MscL from M. tuberculosis (Chang, Spencer, et al., 1998). The expansion of the barrel is achieved by tilting and iris-like motion of the transmembrane helixes M1 (yellow) and M2 (cyan). The existence of a pre-expanded intermediate state was predicted from the kinetic analysis (Sukharev, Sigurdson et al., 1999). The helical structure of S1 domains (red) was inferred from the amphipathic sequence of N-termini, which were unresolved in the crystal structure. While the barrel expands and flattens, the C-terminal helices (purple) remain associated in all conformations.

         

 

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Disulfide cross-links that support the relative positions of helices in the closed (left) and open (right ) conformations.    From Sukharev et al., 2001; Betanzos et al., 2002; and Anishkin et al., 2003.

   

 

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Structure of the C-terminal domains of E. coli MscL modeled after the crystal structure of the 5-fold coiled-coil protein COMP.   The helical interaction is stabilized by apolar interactions  of conserved aliphatic residues inside the bundle (yellow surface), and multiple salt bridges on the periphery. 

     

 

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The molecular dynamic simulation shows that when the ends of linkers are pulled radially to the predicted positions corresponding to the open state, the C-terminal bundle remains stable. From Anishkin et al., 2003. 

         

 

bulletThe model of the open state of MscL with the 'hanging basket' of associated S3 helices. We propose this arrangement makes a pre-filter at the cytoplasmic entrance, excluding high-molecular-weight substances from entering the pore.

                 

 

bulletDetermination of the MscL protein expansion. (A) Models of the closed and open conformations shown with solvent-accessible surfaces predict the mean in-plane expansion of 23.3 nm2. Blue planes represent the boundaries of the hydrocarbon slab. (B) Activation curves measured on multi-channel patches display inflections due to a slight non-homogeneity of channel populations. The initial slope of left-most parts of the curves predicts area and energy change DeltaA=20.44.8 nm2 and DeltaE = 5113 kT as gross thermodynamic parameters for the gating transition (Chiang et al., 2004).
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Gating patterns and analysis of WT MscL and gain-of-function (GOF) mutants. (A) Raw trace with expanded fragment illustrating the substate structure. (B) Amplitude histograms fitted with eleven Gaussian distributions defining probabilities for individual states; the peaks for the closed (C), open (O) and two low-conducting states (S0.13 and S0.22) are shown in color. (C) Plots of probability ratios for the pairs of C, S0.13 and O states as functions of tension.  (D) Spatial and energetic parameters of major substates, extracted from such tension dependencies allow partial reconstruction of gating energy profile for WT and GOF mutants (Anishkin et al., 2005).
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bulletThe crystal structure of the small conductance mechanosensitive channel (MscS) has been an invaluable tool in the search for the gating mechanism, however many functional aspects of the channel remain unsettled. 
The pore constriction and outer chamber of MscS are lined with hydrophobic sidechains  L109, L105, A102, and A98 respectively. Hydration energy estimations, as well as molecular dynamics simulations reveal strong tendency to dewetting. We propose that the crystal structure represents a non-conductive inactivated state of the channel (Anishkin and Sukharev, 2004). Both inactivated and hypothetical closed states of the channel are likely occluded by a "vapor lock".
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bulletMscS displays peculiar channel kinetics characterized by tension-dependent activation, and voltage-dependent inactivation occurring at intermediate tensions (A). Thermodynamic and kinetic analysis of MscS patch-clamp recording allowed us to reconstruct the main features of its energetic landscape in two dimensions representing the "expansion area" and "transferred charge" (B). Based on the extracted expansion and gating charge values, as well as on the pore dewetting simulations we proposed the gating scheme of MscS and currently developing structural models using steered molecular dynamics simulations (C) (Akitake et al., 2005).
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