TY - JOUR
T1 - Cysteine Scanning of MscL Transmembrane Domains Reveals Residues Critical for Mechanosensitive Channel Gating
AU - Levin, Gal
AU - Blount, Paul
N1 - Funding Information:
This work was supported by grants GM61028 and DK60818 from the National Institutes of Health, grant I-1420 of the Welch Foundation, and grant F49620-01-1-0503 of the Air Force Office of Scientific Review.
PY - 2004/5
Y1 - 2004/5
N2 - The mechanosensitive channel of large conductance (MscL), a bacterial channel, is perhaps the best characterized mechanosensitive protein. A structure of the Mycobacterium tuberculosis ortholog has been solved by x-ray crystallography, but details of how the channel gates remain obscure. Here, cysteine scanning was used to identify residues within the transmembrane domains of Escherichia coli MscL that are crucial for normal function. Utilizing genetic screens, we identified several mutations that induced gain-of-function or loss-of-function phenotypes in vivo. Mutants that exhibited the most severe phenotypes were further characterized using electrophysiological techniques and chemical modifications of the substituted cysteines. Our results verify the importance of residues in the putative primary gate in the first transmembrane domain, corroborate other residues previously noted as critical for normal function, and identify new ones. In addition, evaluation of disulfide bridging in native membranes suggests alterations of existing structural models for the "fully closed" state of the channel.
AB - The mechanosensitive channel of large conductance (MscL), a bacterial channel, is perhaps the best characterized mechanosensitive protein. A structure of the Mycobacterium tuberculosis ortholog has been solved by x-ray crystallography, but details of how the channel gates remain obscure. Here, cysteine scanning was used to identify residues within the transmembrane domains of Escherichia coli MscL that are crucial for normal function. Utilizing genetic screens, we identified several mutations that induced gain-of-function or loss-of-function phenotypes in vivo. Mutants that exhibited the most severe phenotypes were further characterized using electrophysiological techniques and chemical modifications of the substituted cysteines. Our results verify the importance of residues in the putative primary gate in the first transmembrane domain, corroborate other residues previously noted as critical for normal function, and identify new ones. In addition, evaluation of disulfide bridging in native membranes suggests alterations of existing structural models for the "fully closed" state of the channel.
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U2 - 10.1016/S0006-3495(04)74338-6
DO - 10.1016/S0006-3495(04)74338-6
M3 - Article
C2 - 15111403
AN - SCOPUS:2142708147
SN - 0006-3495
VL - 86
SP - 2862
EP - 2870
JO - Biophysical journal
JF - Biophysical journal
IS - 5
ER -