Correlating a protein structure with function of a bacterial mechanosensitive channel.
MscL, a mechanosensitive channel found in many bacteria, protects cells from hypotonic shock by reducing intracellular pressure through release of cytoplasmic osmolytes. First isolated from Escherichia coli, this protein has served as a model for how a protein senses and responds to membrane tension. Recently the structure of a functionally uncharacterized MscL homologue from Mycobacterium tuberculosis was solved by x-ray diffraction to a resolution of 3.5 A. Here we demonstrate that the protein forms a functional MscL-like mechanosensitive channel in E. coli membranes and azolectin proteoliposomes. Furthermore, we show that M. tuberculosis MscL crystals, when re-solubilized and reconstituted, yield wild-type channel currents in patch clamp, demonstrating that the protein does not irreversibly change conformation upon crystallization. Finally, we apply functional clues acquired from the E. coli MscL to the M. tuberculosis channel and show a mechanistic correlation between these channels. However, the inability of the M. tuberculosis channel to gate at physiological membrane tensions, demonstrated by in vivo E. coli expression and in vitro reconstitution, suggests that the membrane environment or other additional factors influence the gating of this channel.
Moe
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PC
Department of Physiology, University of Texas-Southwestern Medical Center, Dallas, Texas 75390-9040, USA.
Levin
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Blount
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D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D003461:Crystallography; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010641:Phenotype; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000737:chemistry; D011487:Protein Conformation; D011510:Proteolipids / Q000737:chemistry; D013329:Structure-Activity Relationship; D013997:Time Factors; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; C020485:proteoliposomes; C024990:asolectin