Correlating a protein structure with function of a bacterial mechanosensitive channel

Area of Science:

• Microbiology
• Biophysics
• Structural Biology

Background:

• MscL (mechanosensitive channel of large conductance) is crucial for bacterial survival under hypotonic stress.
• Escherichia coli MscL is a well-studied model for mechanosensitive channel gating.
• A homologous MscL from Mycobacterium tuberculosis was recently structurally characterized.

Purpose of the Study:

• To determine the functional activity of the Mycobacterium tuberculosis MscL homolog.
• To investigate if M. tuberculosis MscL retains function after crystallization and reconstitution.
• To compare the gating mechanisms of M. tuberculosis MscL and E. coli MscL.

Main Methods:

• Functional reconstitution of M. tuberculosis MscL into E. coli membranes and azolectin proteoliposomes.
• Patch-clamp electrophysiology to measure channel activity.
• In vivo expression in E. coli and in vitro reconstitution assays.

Main Results:

• M. tuberculosis MscL forms a functional mechanosensitive channel in reconstituted systems.
• Crystallized M. tuberculosis MscL, when re-solubilized, exhibits wild-type channel currents.
• Mechanistic correlations were observed between M. tuberculosis MscL and E. coli MscL.
• M. tuberculosis MscL failed to gate at physiological membrane tensions in vivo and in vitro.

Conclusions:

• M. tuberculosis MscL is a functional mechanosensitive channel, but its gating is less sensitive than E. coli MscL.
• The bacterial membrane environment or other factors likely modulate M. tuberculosis MscL gating.
• Structural information of M. tuberculosis MscL can be correlated with functional data from E. coli MscL.