In vitro synthesis and oligomerization of the mechanosensitive channel of large conductance, MscL, into a functional ion channel.
Elucidation of high-resolution structures of integral membrane proteins is drastically lagging behind that of cytoplasmic proteins. In vitro synthesis and insertion of membrane proteins into synthetic membranes could circumvent bottlenecks associated with the overexpression of membrane proteins, producing sufficient membrane-inserted, correctly folded protein for structural studies. Using the mechanosensitive channel of large conductance, MscL, as a model protein we show that in vitro synthesized MscL inserts into YidC-containing proteoliposomes and oligomerizes to form a homopentamer. Using planar membrane bilayers, we show that MscL forms functional ion channels capable of ion transport. These data demonstrate that membrane insertion of MscL is YidC mediated, whereas subsequent oligomerization into a functional homopentamer is a spontaneous event.
Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Claire E
CE
Price
Armagan
A
Kocer
Stefan
S
Kol
Jan Pieter
JP
van der Berg
Arnold J M
AJ
Driessen
D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015640:Ion Channel Gating; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017136:Ion Transport; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D040542:Mechanotransduction, Cellular; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D055503:Protein Multimerization; D000069816:SEC Translocation Channels
D029968:Escherichia coli Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; C086596:MscL protein, E coli; D000069816:SEC Translocation Channels; C412443:YIDC protein, E coli