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

Abstract

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.

Correct Folding of the β-Barrel of the Human Membrane Protein VDAC Requires a Lipid Bilayer

“…Clearly, hVDAC1 forms a less stable three-dimensional fold than bacterial OMPs, since the secondary structure and resistance to proteolysis depend strongly on the environment of hVDAC1, whether it is a detergent micelle or a lipid bilayer. Proteolysis of bacterial OMPs in detergent micelles does neither result in multiple fragments nor is proteolysis complete within 1 h. 15 The reduced stability of hVDAC1 in detergent is also evident by an increased pH sensitivity of the hVDAC1 secondary structure in micelles compared to bilayers (Figure 4(b)). Our observations suggest that hVDAC1 is present in partially folded/denatured form even in milder detergents than SDS.…”

“…10,12,13 Bacterial porins of similar size like OmpF and FomA, contain only ∼ 2 to 5% helical structure according to X-ray crystallography (2OMF) 14 or CD spectroscopy. 15 The majority of studies on the structure of VDAC have been performed in detergent micelles, while VDAC function was assayed in lipid bilayers. 9 Despite the necessity of proteinaceous folding factors in cells, several biochemical studies have shown that unfolded bacterial OMPs in 8 M urea can spontaneously fold into detergent micelles to their active form upon urea dilution (for a review, see Buchanan 16 ).…”

“…9 Despite the necessity of proteinaceous folding factors in cells, several biochemical studies have shown that unfolded bacterial OMPs in 8 M urea can spontaneously fold into detergent micelles to their active form upon urea dilution (for a review, see Buchanan 16 ). The outer membrane proteins OmpA, 17,18 OmpF 19 of E. coli and FomA of Fusobacterium nucleatum 15 are the only β-barrel membrane proteins, for which folding of the urea-unfolded forms into lipid bilayers has been demonstrated, although attempts were also made with several other OMPs. Detergent micelles dissociate and reform fast, while lipid bilayers and biological membranes are tightly packed.…”

“…15 Upon addition of hVDAC1 in LDAO, a stepwise increase of the current across the lipid bilayer was observed (Figure 3(a), inset) and 50% of the corresponding channel events had a conductance of 4.5(± 0.5) nS, indicating insertion of single channels. For a single channel opening, a The composition of the secondary structure of hVDAC1 in the different samples was analyzed as described in the legend to Table 1.…”

“…Analysis II was performed on hVDAC1 that was first solubilized in LDAO and then reconstituted into diC 12:0 PC (the CD spectrum is shown in Figure 1 3,5,7,9,12,14,16), and in diC 12:0 PC (lanes 4,6,8,10,13,15,17), indicate formation of a single fragment of ∼ 26 to 27 kDa size in diC 12:0 PC bilayers that is present after 240 min of proteolysis. In LDAO, hVDAC1 is first cleaved into two major fragments of ∼ 26 to 27 kDa and of ∼ 24 kDa.…”