Open-state disulfide crosslinking between Mycobacterium tuberculosis mechanosensitive channel subunits.
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.
Shapovalov
George
G
Division of Biology, California Institute of Technology, Pasadena, 91125, USA.
Bass
Randal
R
Rees
Douglas C
DC
Lester
Henry A
HA
D001665:Binding Sites; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000235:genetics / Q000502:physiology; D004220:Disulfides / Q000737:chemistry*; D046911:Macromolecular Substances; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology; D009154:Mutation; D010812:Physical Stimulation / Q000379:methods; D011312:Pressure; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013104:Spheroplasts / Q000737:chemistry* / Q000502:physiology*
D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D003545:Cysteine