The Characterization and Structure of Mechanosensitive Channels of Small Conductance

Citation

Poon, Yan Shuen (2008) The Characterization and Structure of Mechanosensitive Channels of Small Conductance. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/EBZ2-5D39. https://resolver.caltech.edu/CaltechETD:etd-05292008-144531

Abstract

This thesis describes the investigation of the mechanosensitive channel of small conductance (MscS). The Escherichia coli MscS structure shows a homoheptameric ion channel with each monomer consisting of 3 transmembrane (TM) helices leading into the cytoplasmic domain. TM3 from each of the 7 subunits form the pore that opens up into a cytoplasmic cage, thought to be a molecular sieve. In order to further knowledge regarding the mechanism behind bacterial mechanosensation, several approaches were employed. Homologs from organisms indigenous to environments with temperatures ranging from 30°C to 95°C were cloned, expressed, and characterized. Chimeras were constructed between MscS homologs from 6 hyperthermophilic organisms and E. coli MscS, exchanging the transmembrane and cytoplasmic domains. Each protein is engineered with an N-terminal His6 tag and a C-terminal FLAG epitope. 4 homologs were found to be cytotoxic while 3 chimeras failed to rescue the osmotic-shock sensitivity of an E. coli mutant strain lacking endogenous mechanosensitive ion channel activity.

Studies revolving around the various homologs and chimeras led to the crystallization of the Helicobacter pylori MscS homolog. H. pylori MscS shares approximately 35% in sequence identity and was crystallized in space group P212121 with cell dimensions a = 123.0 A, b = 147.4 A, c = 179.4 A. Diffraction data was collected at the Stanford Synchrotron Radiation Laboratory at beamline 12-2 to 4.75 A resolution. A molecular replacement solution was achieved using the program PHASER with the E. coli MscS model (PDB: 2OAU). The refined structure shows a similar overall topology with E. coli MscS demonstrating a homoheptameric channel. The cytoplasmic domain maintains the same structure while TM1 and TM2 display slightly tighter packing with the pore. The structure of the pore suggests that H. pylori and E. coli MscS are crystallized in the same state.

Thesis Files