Refolding of Escherichia coli outer membrane protein F in detergent creates LPS-free trimers and asymmetric dimers

Abstract

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.

Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I

“…Mesophilic proteins with T m values above 45°C are generally considered to be thermostable (70,71); all of the proteins studied herein met this criterion. Of note, the T m for OmpF is in close agreement with published values determined by both CD and differential scanning calorimetry (72). Each curve shows a sharp unfolding transition, indicating a two-state thermal unfolding, all of which were irreversible upon cooling from 90 set of experiments, we examined TprC C folded in DDM.…”

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

“…OmpF trimers show identical structural and electrophysiological properties to those that folded in vivo in the presence of LPS in the OM (17,18).…”

“…Here we provide support for these observations by showing that the successful in vivo OM biogenesis of OmpF requires the presence of an intact LPS B site at the periphery of the trimer. By contrast, the successful production of functional LPS-free OmpF by folding into detergent micelles suggested that LPS was not an essential component for OmpF in vitro (17,47). OMP biogenesis is mediated by the BAM complex, which accepts unfolded proteins from periplasmic chaperones (48) and mediates the formation of β-barrels in the OM via a mechanism that is not yet well-understood (49)(50)(51).…”

“…3 A-C). To test whether site B mutants could still form trimers in vitro, we purified the most radical mutants (lysine/ arginine-to-glutamate substitutions, which reverse the charge of the sites) as inclusion bodies by expressing them without signal sequences (17). The inclusion bodies were solubilized in urea and ran on SDS/PAGE as monomers with slightly altered mobilities (Fig.…”

Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi

“…Refolding of porins from inclusion bodies (IBs) and their structure determination was successful in the case of Rhodopseudomonas blastica porin (Schmid et al, 1996) and OpCA from Neisseria meningitidis (Prince et al, 2001) where the refolded proteins showed structural similarity to their native structures. OmpF from E. coli had been overexpressed and refolded in the presence of detergents (Miedema et al, 2004;Visudtiphole et al, 2005). However, this is the first report of crystallisation and structure determination of in vitro refolded OmpF from a human pathogen.…”