TY - JOUR
T1 - Disulfide trapping the mechanosensitive channel MscL into a gating-transition state
AU - Iscla, Irene
AU - Levin, Gal
AU - Wray, Robin
AU - Blount, Paul
N1 - Funding Information:
This work was supported by grant I-1420 of the Welch Foundation, grant FA9550-05-1-0073 of the Air Force Office of Scientific Review, grant 0655012Y of the American Heart Association-Texas Affiliate, and grant GM61028 from the National Institutes of Health.
PY - 2007/2
Y1 - 2007/2
N2 - The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock, and is to date the best characterized mechanosensitive channel. The N-terminal region of the protein has been shown to be critical for function by random, site-directed, and deletion mutagenesis, yet is structurally poorly understood. One model proposes that the extreme N-termini form a cluster of amphipathic helices that serves as a cytoplasmic second gate, separated from the pore-forming transmembrane domain by a "linker". Here, we have utilized cysteine trapping of single-cysteine mutated channels to determine the proximity, within the homopentameric complex, of residues within and just peripheral to this proposed linker. Our results indicate that all residues in this region can form disulfide bridges, and that the percentage of dimers increases when the channel is gated in vivo. Functional studies suggest that oxidation traps one of these mutated channels, N15C, into a gating-transition state that retains the capacity to obtain both fully open and closed states. The data are not easily explained by current models for the smooth transition from closed-to-open states, but predict that an asymmetric movement of one or more of the subunits commonly occurs upon gating.
AB - The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock, and is to date the best characterized mechanosensitive channel. The N-terminal region of the protein has been shown to be critical for function by random, site-directed, and deletion mutagenesis, yet is structurally poorly understood. One model proposes that the extreme N-termini form a cluster of amphipathic helices that serves as a cytoplasmic second gate, separated from the pore-forming transmembrane domain by a "linker". Here, we have utilized cysteine trapping of single-cysteine mutated channels to determine the proximity, within the homopentameric complex, of residues within and just peripheral to this proposed linker. Our results indicate that all residues in this region can form disulfide bridges, and that the percentage of dimers increases when the channel is gated in vivo. Functional studies suggest that oxidation traps one of these mutated channels, N15C, into a gating-transition state that retains the capacity to obtain both fully open and closed states. The data are not easily explained by current models for the smooth transition from closed-to-open states, but predict that an asymmetric movement of one or more of the subunits commonly occurs upon gating.
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U2 - 10.1529/biophysj.106.090316
DO - 10.1529/biophysj.106.090316
M3 - Article
C2 - 17114217
AN - SCOPUS:33846849245
SN - 0006-3495
VL - 92
SP - 1224
EP - 1232
JO - Biophysical journal
JF - Biophysical journal
IS - 4
ER -