Refolding of Escherichia coli outer membrane protein F in detergent creates LPS-free trimers and asymmetric dimers.
The Escherichia coli OmpF (outer-membrane protein F; matrix porin) is a homotrimeric beta-barrel and a member of the bacterial porin superfamily. It is the best characterized porin protein, but has resisted attempts to refold it efficiently in vitro. In the present paper, we report the discovery of detergent-based folding conditions, including dodecylglucoside, which can create pure samples of trimeric OmpF. Whereas outer membrane LPS (lipopolysaccharide) is clearly required for in vivo folding, the artificially refolded and LPS-free trimer has properties identical with those of the outer-membrane-derived form. Thus LPS is not required either for in vitro folding or for structural integrity. Dimeric forms of OmpF have been observed in vivo and are proposed to be folding intermediates. In vitro, dimers occur transiently in refolding of trimeric OmpF and, in the presence of dodecylmaltoside, pure dimer can be prepared. This form has less beta-structure by CD and shows lower thermal stability than the trimer. Study of these proteins at the single-molecule level is possible because each OmpF subunit forms a distinct ion channel. Whereas each trimer contains three channels of equal conductance, each dimer always contains two distinct channel sizes. This provides clear evidence that the two otherwise identical monomers adopt different structures in the dimer and indicates that the asymmetric interaction, characteristic of C3 symmetry, is formed at the dimer stage. This asymmetric dimer may be generally relevant to the folding of oligomeric proteins with odd numbers of subunits such as aspartate transcarbamoylase.
Visudtiphole
Virak
V
Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle NE1 7RU, UK.
Thomas
Matthew B
MB
Chalton
David A
DA
Lakey
Jeremy H
JH
D003902:Detergents / Q000737:chemistry / Q000494:pharmacology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000737:chemistry*; D008958:Models, Molecular; D018272:Porins / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000187:drug effects
D003902:Detergents; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; C109237:OmpF protein; D018272:Porins