Engineering covalent oligomers of the mechanosensitive channel of large conductance from Escherichia coli with native conductance and gating characteristics

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Abstract
To obtain a gene construct for making single substitutions per channel and to determine the quaternary structure of the mechanosensitive channel MscL from Escherichia coli, covalent oligomers (monomer to hexamer) were engineered by gene fusion; up to six copies of the mscL gene were fused in tandem. All the multimeric tandem constructs yielded functional channels with wild‐type conductance and dwell times. Importantly, only the covalent pentamer opened at the same relative pressure (compared to the pressure required to open MscS) as the wild‐type MscL channel. The in vivo data strongly suggest that pentameric MscL represents the functional state of the channel.