Structural characterization of the fusobacterial non-specific porin FomA suggests a 14-stranded topology, unlike the classical porins

Puntervoll, Pål; Ruud, Morten; Bruseth, Live J.; Kleivdal, Hans; Högh, Bente T.; Benz, Roland; Jensen, Harald B.

doi:10.1099/00221287-148-11-3395

Cited by 29 publications

(31 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…24 The structure of FomA has not yet been solved, but recent circular dichroism (CD) spectroscopy and topology prediction suggested a 14-stranded transmembrane b-barrel. 25 FomA forms voltage-dependent diffusion channels, 25,26 which have a conductivity of w1.2 nS, i.e. much greater than w250-320 pS reported for the large open state of OmpA, 27 which is in agreement with a larger size of the transmembrane domain of FomA.…”

Section: Introductionsupporting

confidence: 68%

“…This property was first described for OmpA of E. coli, 28 and later for other OMPs such as OmpG, 11 FhuA, 29 and FomA. 25,26 On SDS-polyacrylamide gels, FomA migrates at 37 kDa in native form when isolated from membranes and at 40 kDa when denatured either by heat or by urea. 25 To first investigate the conditions required for the folding of FomA, we examined the electrophoretic mobility of FomA in unfolded form in urea, after denaturant dilution in aqueous solution, and also after incubation with detergent micelles or lipid bilayers, which in previous studies induced folding of OmpA.…”

Section: Resultsmentioning

confidence: 99%

“…13,15,21 To determine whether the refolded 37 kDa form of FomA had inserted into lipid bilayers or detergent micelles, we performed digestion experiments with trypsin, because we expected that micelle-or membrane-inserted parts of the polypeptide chain of FomA would be protected against degradation as observed previously for FomA isolated in native form from cell envelopes. 25 Figure 1(b) shows that both, denatured (lane 1) and water-collapsed (lane 3) FomA were completely digested by trypsin (lanes 2 and 4), since no band could be observed on SDS-PAGE after 2 h of incubation of FomA with trypsin at 37 8C. In detergent micelles (data not shown) and lipid bilayers (Figure 1(b), lanes 5 to 12), most of the polypeptide chain of FomA was protected against trypsin digestion, which led to a 34 kDa fragment similar as in previous experiments with FomA isolated in native form in membranes.…”

Section: Resultsmentioning

confidence: 99%

“…25,26 On SDS-polyacrylamide gels, FomA migrates at 37 kDa in native form when isolated from membranes and at 40 kDa when denatured either by heat or by urea. 25 To first investigate the conditions required for the folding of FomA, we examined the electrophoretic mobility of FomA in unfolded form in urea, after denaturant dilution in aqueous solution, and also after incubation with detergent micelles or lipid bilayers, which in previous studies induced folding of OmpA. 7,16,17 We expected that successful folding of FomA should change the electrophoretic mobility from 40 kDa of the unfolded form to 37 kDa of the native form as seen in previous studies on OmpA, which migrated at 35 kDa in unfolded form and at 30 kDa in refolded, functionally active form.…”

Section: Resultsmentioning

confidence: 99%

See 3 more Smart Citations

The Major Outer Membrane Protein of Fusobacterium nucleatum (FomA) Folds and Inserts into Lipid Bilayers via Parallel Folding Pathways

Pocanschi

Apell

Puntervoll

et al. 2006

Journal of Molecular Biology

Self Cite

105

View full text Add to dashboard Cite

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltagedependent general diffusion porin. The transmembrane domain of FomA forms a b-barrel that is predicted to consist of 14 b-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded b-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a b-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.

show abstract

Section: Introductionsupporting

confidence: 68%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

The Major Outer Membrane Protein of Fusobacterium nucleatum (FomA) Folds and Inserts into Lipid Bilayers via Parallel Folding Pathways

Pocanschi

Apell

Puntervoll

et al. 2006

Journal of Molecular Biology

Self Cite

105

View full text Add to dashboard Cite

show abstract

“…1 for some examples). Predicted 14-stranded b-barrel membrane proteins are FomA [15] and OmpG [16,17]. Monomers (for example OmpA, FhuA), dimers (OmPlA) and trimers (OmpF, PhoE) are known.…”

Section: Introductionmentioning

confidence: 99%

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

Kleinschmidt¹

2003

Cellular and Molecular Life Sciences (CMLS)

View full text Add to dashboard Cite

Abstract. The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded b-barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the b-barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of b-sheet secondary structure and b-barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i.e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of b-barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates.

show abstract

One-Step Purification and Porin Transport Activity of the Major Outer Membrane Proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis

Kattner

Pfennig

Massi

et al. 2015

Appl Biochem Biotechnol

View full text Add to dashboard Cite

Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.

show abstract

Structural characterization of the fusobacterial non-specific porin FomA suggests a 14-stranded topology, unlike the classical porins

Cited by 29 publications

References 37 publications

The Major Outer Membrane Protein of Fusobacterium nucleatum (FomA) Folds and Inserts into Lipid Bilayers via Parallel Folding Pathways

The Major Outer Membrane Protein of Fusobacterium nucleatum (FomA) Folds and Inserts into Lipid Bilayers via Parallel Folding Pathways

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

One-Step Purification and Porin Transport Activity of the Major Outer Membrane Proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis

Contact Info

Product

Resources

About