Open-State Disulfide Crosslinking between Mycobacterium tuberculosis Mechanosensitive Channel Subunits
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 β-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 ≥100 s. These signature-events were abolished by 100 mM β-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 α-carbons of adjacent 15C side chains within 3.6–6.8 Å. Such asymmetric structures must be considered in models of TB-MscL gating.
Additional Information
© 2003 by the Biophysical Society. Submitted August 30, 2002, and accepted for publication December 9, 2002. We thank Sergei Sukharev and Paul Blount for introducing us to MscL electrophysiology, Josh Maurer for mutants and cells, and Ido Braslavsky, Donald Elmore, Steve Quake, Gerd Kochendoerfer, and Dennis Dougherty for valuable discussion. This research was supported by a grant from the National Institutes of Health (GM-062532), by a Burroughs-Wellcome Fund Computation and Molecular Biology Fellowship to George Shapovalov, and by an National Research Service Award to Randal Bass (GM-020705).Attached Files
Published - shapovalov_2003.pdf
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Additional details
- PMCID
- PMC1302802
- Eprint ID
- 52788
- Resolver ID
- CaltechAUTHORS:20141212-145345633
- NIH
- GM-062532
- Burroughs-Wellcome Fund
- NIH
- GM-020705
- Created
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2014-12-22Created from EPrint's datestamp field
- Updated
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2023-10-18Created from EPrint's last_modified field