Open-state disulfide crosslinking between Mycobacterium tuberculosis mechanosensitive channel subunits |
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| Authors: | Shapovalov George Bass Randal Rees Douglas C Lester Henry A |
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| Affiliation: | Division of Biology, California Institute of Technology, Pasadena, 91125, USA. |
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| Abstract: | The mechanosensitive channel of large conductance from Mycobacterium tuberculosis (Tb-MscL) was subjected to cysteine-scanning mutagenesis at several residues in the M1 region. The V15C channel displayed disulfide crosslinking in air, but not in the presence of 100 mM beta-mercaptoethanol. In single-channel experiments, the V15C channel was more sensitive to tension than was wild-type Tb-MscL. In air, Tb-MscL V15C occasionally displayed signature-events: at constant tension, there was first a sojourn in the highest conductance open state, then a series of transitions to substates. During a signature-event, these transitions do not appear to be reversible. Some sojourns in the lower conductance states lasted for > or =100 s. These signature-events were abolished by 100 mM beta-mercaptoethanol and did not occur in a cysteineless gain-of-function mutant, suggesting that the signature-events represent disulfide crosslinking between channel subunits. We conclude that the crosslinking occurs during an open state during asymmetric sojourns that bring the alpha-carbons of adjacent 15C side chains within 3.6-6.8 A. Such asymmetric structures must be considered in models of TB-MscL gating. |
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