TY - JOUR
T1 - Single residue substitutions that change the gating properties of a mechanosensitive channel in Escherichia coli
AU - Blount, Paul
AU - Sukharev, Sergei I.
AU - Schroeder, Matthew J.
AU - Nagle, Scott K.
AU - Kung, Ching
PY - 1996/10/15
Y1 - 1996/10/15
N2 - 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.
AB - 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.
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U2 - 10.1073/pnas.93.21.11652
DO - 10.1073/pnas.93.21.11652
M3 - Article
C2 - 8876191
AN - SCOPUS:0029909197
SN - 0027-8424
VL - 93
SP - 11652
EP - 11657
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 21
ER -