Analysis of the function and stability of the mechanosensitive channel of small conductance (MscS)

When Escherichia coli cells are grown in a highly osmotic environment the cell accumulates compatible solutes to stop the outflow of water from the cytoplasm due to osmosis. If this same cell were diluted to low osmolarity media water moves into the cell to dilute the internal contents this dramatically increases the internal pressure of the cell. This increase in pressure of the cytoplasm must be released to avoid rupturing the cell membrane. The mechanosensitive channels sense the tension in the membrane and respond to this increase in cytoplasmic pressure. The mechanosensitive channel of small conductance (MscS) is one such channel and it is essential for the survival of E. coli from hypoosmotic shock. In this thesis I have addressed three questions. Firstly I examined the population affects of hypo osmotic shock on E. coli. Secondly I examined the functional role of the N-terminal, periplasmic region, of Ms cS. This region was not resolved in the crystal structure but previous work has suggested that this region may play a role in MscS channel (heptamer) stability. In the third section I further examined the stability of the MscS channel. This was done by developing two techniques to examine protein stability of the protein complex. I have shown that there are several populations present after hypoosmotic shock, caused by heterogeneity present in clonal populations. I have also shown that the N-terminus of MscS may play a role in channel stability but it is not essential in this role. I have also shown that the N-terminus may cover the pore of the channel, as indicated by protein fluorescence. Thirdly I have successfully developed a technique to examine the melting temperature of mutant MscS heptameric proteins, using the dye SYPRO orange and thermal shift assay based around a qRT-PCR machine. This thesis has addressed several fundamental questions concerned with the MscS channel .and has lead to several advances in our understanding about both population dynamics after hypoosmotic shock and about how protein complexes remain stable in the membrane.EThOS - Electronic Theses Online ServiceGBUnited Kingdo