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Open-State Disulfide Crosslinking between Mycobacterium tuberculosis Mechanosensitive Channel Subunits

George Shapovalov^*,, Randal Bass, Douglas C. Rees^, and Henry A. Lester^*

^* Division of Biology; Division of Physics, Mathematics, and Astronomy; Division of Chemistry and Chemical Engineering; and Howard Hughes Medical Institute; California Institute of Technology, Pasadena, California 91125

Correspondence: Address reprint requests to Henry A. Lester, Div. of Biology 156-29, California Institute of Technology, Pasadena, CA 91125. Tel.: 626-395-4946; Fax: 626-564-8709; E-mail: lester{at}caltech.edu.

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 ß-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 ß-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 -carbons of adjacent 15C side chains within 3.6–6.8 Å. Such asymmetric structures must be considered in models of TB-MscL gating.

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