Determining Structural Transitions That Occur Upon Gating a Bacterial Mechanosensitive Channel



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Essentially all bacteria, including pathogens, must be able to rapidly adapt to changing environments, specifically a rapid decrease in osmotic environment which can result in major trauma to the cell. The mechanosensitive channel of large conductance (MscL) is one of a handful of channels that responds to tension in the membrane and acts as a lifesaving mechanism for the cell in instances of osmotic down shock. Solutes are jettisoned before the cell explodes due to internal pressures. The goal of this project was to further define the mechanism of opening of the MscL channel. I used two approaches to solve this problem. The first utilized the Substituted-Cysteine Accessibility Method (SCAM). Specific amino acids are replaced with a cysteine residue and the ability of that cysteine to react with a substrate is assessed. A cysteine library of the transmembrane domains of E. coli MscL was created. I screened the mutants for their ability to react with MTSET before and after shock using whole-cell physiological assays. This in vivo SCAM study gave support for a clockwise rotation of TM1 predicted in another model, and defined a number of residues that appear to constitute the pore of the open E. coli MscL channel. Furthermore, the precise manner in which the channel activity was modified by the MTSET reagent was then determined by examining a select number of residues using electrophysiology. In this way, the transition from closed to open states could be examined. The data presented confirm many of our previous predictions as well as give new insight into the structural transitions that occur upon gating. The second approach to define opening utilized functional differences between homologues that have relatively similar sequences. For instance, the E. coli and M. tuberculosis MscL proteins are similar in sequence. However, they exhibit different sensitivities to pressure both in vivo and in vitro. Another homologue, found in S. aureus, exhibits faster kinetics and a different conductance. The chimeras constructed between E. coli and M. tuberculosis and E. coli and S. aureus MscLs have given insight into structural domains that can alter channel threshold tension, kinetics and conductance.