Exploring the Dynamics of Mechanosensitive Channels Utilizing a Post Translational Modification Approach

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It is essential for all living organisms to possess the ability to sense and respond to changes in their environment. The ability to detect external and internal mechanical force is very important as well. The detection of mechanical forces underlies the ability to sense touch, gravity, blood pressure, and changes in osmolarity. The bacterial mechanosensitive channel of large conductance (MscL) and the mechanosensitive channel of small conductance (MscS), from E. coli, have been the most studied mechanosensors to date. These channels coordinate a response to rapid changes in osmolarity to serve as emergency release valves during an osmotic downshock (sudden change to a lower osmotic environment). Crystal structures have been solved for homologues of each of these channels. In addition, patch clamp of giant bacterial spheroplasts has allowed for the study of channel activities in native membranes, and both channels have been functionally reconstituted into membranes and studied by electrophysiological single-channel analyses. However, even though well studied, it is still unclear how specific regions of the protein interact with the membrane, and how these interactions change upon channel gating. Here we hypothesize that during channel gating specific residues of MscL change their local environment. We investigate this hypothesis by using a post-translational modification approach to determine how changing hydrophobicity or charge at specific locations influences gating. Briefly, we used MscL channels with specific residues in the linker between the second transmembrane region (TM2) and the cytoplasmic α-helical bundle changed to cysteine. Changing the individual amino acids to cysteine allows us to use Methanethiosulfonate (MTS) sulfhydryl reagents that can form disulfide bonds with cysteines to change the properties (charge or hydrophobicity) of specific residues. We then use the double knockout strain, ∆MscL/∆MscS, which is osmotically fragile and has a reduced viability when osmotically downshocked. These cells are osmotically downshocked in the presence or absence of different hydrophobic or charged MTS sulfhydryl reagents with different affinities for membrane or membrane/water interface environment, or that possess different charges. The assumption is that the channel changes conformation, and lipid interactions, upon gating. By changing the hydropathy of residues at specific locations, we drag a portion of the protein into a different environment, thus increasing or decreasing the probability of opening. This leads to a channel that opens too frequently or one that fails to open and this can be assessed by measuring changes in cell viability. Here we focused on the specific MscL residues F83-E107 to expand previous work performed on this region of the MscL protein. While the bulk of residues investigated here did not reach a high enough threshold to warrant further investigation, a few of the modifications are of interest. To investigate channels across channel families it would be of interest to use this approach for identification of residues that are important for MscS channel gating. Towards this end, I have generated several cysteine mutations in the transmembrane linker regions for MscS. This will allow similar and more extensive studies to be conducted with MscS to determine if the results observed for MscL are general principles observed in the unrelated MscS channel, or if there are variations between the two mechanosensitive channel families.

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