The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways.
Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-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 beta-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 beta-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.
Pocanschi
Cosmin L
CL
Fachbereich Biologie, Universität Konstanz, D-78457 Konstanz, Germany.
Apell
Hans-Jürgen
HJ
Puntervoll
Pål
P
Høgh
Bente
B
Jensen
Harald B
HB
Welte
Wolfram
W
Kleinschmidt
Jörg H
JH
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D016967:Fusobacterium nucleatum / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010713:Phosphatidylcholines / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; C083760:fomA protein, Fusobacterium; C015132:1,2-didecanoylphosphatidylcholine; D011189:Potassium Chloride; D014508:Urea; C017251:1,2-oleoylphosphatidylcholine