Structural and functional analysis of the Escherichia coli small mechanosensitive channel MscS
Mechanosensitive (MS) channels are found in all levels of organisms, and function as transducers in mechanical force pathways. These channels are involved in different aspects of cellular function, in Escherichia coli MS channels are important to maintain cell integrity during hypoosmotic shock conditions. This type of shock is produced when the external environment is more dilute than the internal concentration of the bacterial cell. MS channels gate at this stage and release ions and metabolites to the environment, which decreases the internal pressure and prevents the lysis of the cell. Genetic and physiological studies have determined 3 main classes of MS channel in E. coli, MscL, MscS and MscM. The largest type of channel, MscL has been thoroughly investigated, and with the generation of the crystal structure, gating theories have started to evolve. However there is very little known about the smaller channel, MscS. This project aimed to investigate the structure and function of the MscS channel using mutagenic strategies and physiological assays to understand the implications of the mutations created. Two new protocols have been devised to aid with analysis of mutants - an assay to aid with the identification of GOF mutations, and protein cross-linking assay that indicates conformational change within the protein. Important mutations have been found that help identify the roles of the different regions of the channel. Mutations in TM3 have illustrated the function of this helix as the pore-forming region. Substitutions within TM1 and 2 have demonstrated the importance of the voltage sensing elements and the packing of the helices external to the pore region. Finally, the cytoplasmic domain was also studied and shown to be integral to the gating process. Using current ideas and literature, a gating mechanism can be suggested.