Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli.
MscL is a channel that opens a large pore in the Escherichia coli cytoplasmic membrane in response to mechanical stress. Previously, we highly enriched the MscL protein by using patch clamp as a functional assay and cloned the corresponding gene. The predicted protein contains a largely hydrophobic core spanning two-thirds of the molecule and a more hydrophilic carboxyl terminal tail. Because MscL had no homology to characterized proteins, it was impossible to predict functional regions of the protein by simple inspection. Here, by mutagenesis, we have searched for functionally important regions of this molecule. We show that a short deletion from the amino terminus (3 amino acids), and a larger deletion of 27 amino acids from the carboxyl terminus of this protein, had little if any effect in channel properties. We have thus narrowed the search of the core mechanosensitive mechanism to 106 residues of this 136-amino acid protein. In contrast, single residue substitutions of a lysine in the putative first transmembrane domain or a glutamine in the periplasmic loop caused pronounced shifts in the mechano-sensitivity curves and/or large changes in the kinetics of channel gating, suggesting that the conformational structure in these regions is critical for normal mechanosensitive channel gating.
Blount
P
P
Laboratory of Molecular Biology, University of Wisconsin, Madison 53706, USA.
Sukharev
S I
SI
Schroeder
M J
MJ
Nagle
S K
SK
Kung
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000502:physiology; D001483:Base Sequence; D002462:Cell Membrane / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D015640:Ion Channel Gating; D007473:Ion Channels / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017354:Point Mutation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; D046911:Macromolecular Substances; C086596:MscL protein, E coli; D011993:Recombinant Fusion Proteins