Out but not in: the large transmembrane β-barrel protein FhuA unfolds but cannot refold via β-hairpins.

How transmembrane β-barrel proteins insert and fold into membranes and by which factors they destabilize, unfold, and misfold represents a field of intense studies. Here, we use single-molecule force spectroscopy to characterize the un- and refolding of the ferric hydroxamate uptake receptor (FhuA), which is one of the largest β-barrel proteins of the outer membrane of Escherichia coli. Applied to mechanical stress, FhuA undergoes a complex unfolding pathway in which each of the 11 β-hairpins unfolds one after the other until the entire β-barrel has unfolded. Once unfolded and relaxed, the FhuA polypeptide cannot fold back into the lipid membrane and adopts various misfolded conformations. Such misfolding is in contrast to the reversible refolding behavior of much smaller β-barrel outer membrane proteins OmpA and OmpG that occurs at similar experimental conditions. The results suggest that large β-barrel proteins that show more complex (un-)folding pathways require cofactors for proper insertion and folding into the membrane.

[1]  Kai Simons,et al.  Cell membranes: the lipid perspective. , 2011, Structure.

[2]  L. Tamm,et al.  Folding and assembly of β-barrel membrane proteins , 2004 .

[3]  J. Bowie.  Solving the membrane protein folding problem , 2005, Nature.

[4]  G. Heijne,et al.  Genome‐wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms , 1998, Protein science : a publication of the Protein Society.

[5]  G. Schulz,et al.  High-resolution structure of the OmpA membrane domain. , 2000, Journal of molecular biology.

[6]  K. Dill,et al.  From Levinthal to pathways to funnels , 1997, Nature Structural Biology.

[7]  A. Engel,et al.  Reference-free alignment and sorting of single-molecule force spectroscopy data. , 2012, Biophysical journal.

[8]  Andreas Engel,et al.  Structure and mechanics of membrane proteins. , 2008, Annual review of biochemistry.

[9]  J. Rosenbusch,et al.  Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. , 1998, FEMS microbiology letters.

[10]  D. Engelman.  Membranes are more mosaic than fluid , 2005, Nature.

[11]  S. Buchanan,et al.  Beta-barrel proteins from bacterial outer membranes: structure, function and refolding. , 1999, Current opinion in structural biology.

[12]  Daniel J. Muller,et al.  Imaging and quantifying chemical and physical properties of native proteins at molecular resolution by force-volume AFM. , 2011, Angewandte Chemie.

[13]  K. Diederichs,et al.  An internal affinity‐tag for purification and crystallization of the siderophore receptor fhua, integral outer membrane protein from escherichia coli K‐12 , 1998, Protein science : a publication of the Protein Society.

[14]  D. Tobias,et al.  Assembly and Stability of α-Helical Membrane Proteins. , 2012, Soft matter.

[15]  Kai Simons,et al.  Lipid Rafts As a Membrane-Organizing Principle , 2010, Science.

[16]  Daniel J. Muller,et al.  Observing folding pathways and kinetics of a single sodium-proton antiporter from Escherichia coli. , 2006, Journal of molecular biology.

[17]  S. Radford,et al.  The transition state for folding of an outer membrane protein , 2010, Proceedings of the National Academy of Sciences.

[18]  Robert T Sauer,et al.  AAA+ proteases: ATP-fueled machines of protein destruction. , 2011, Annual review of biochemistry.

[19]  Daniel J Müller,et al.  Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy. , 2004, Journal of molecular biology.

[20]  D. Müller,et al.  The transmembrane protein KpOmpA anchoring the outer membrane of Klebsiella pneumoniae unfolds and refolds in response to tensile load. , 2012, Structure.

[21]  Daniel J Müller,et al.  Atomic force microscopy and spectroscopy of native membrane proteins , 2007, Nature Protocols.

[22]  Daniel J. Muller,et al.  pH-dependent interactions guide the folding and gate the transmembrane pore of the beta-barrel membrane protein OmpG. , 2010, Journal of molecular biology.

[23]  Daniel J Müller,et al.  Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy. , 2007, Annual review of biophysics and biomolecular structure.

[24]  S. White,et al.  Membrane protein folding and stability: physical principles. , 1999, Annual review of biophysics and biomolecular structure.

[25]  A. Telser.  Molecular Biology of the Cell, 4th Edition , 2002 .

[26]  J. Turnbull,et al.  Ligand‐induced conformational change in the ferrichrome–iron receptor of Escherichia coli K‐12 , 1996, Molecular microbiology.

[27]  T. Silhavy,et al.  β-Barrel membrane protein assembly by the Bam complex. , 2011, Annual review of biochemistry.

[28]  Daniel J. Muller,et al.  Bacteriorhodopsin folds into the membrane against an external force. , 2006, Journal of molecular biology.

[29]  Daniel J. Muller,et al.  Dual energy landscape: The functional state of the β‐barrel outer membrane protein G molds its unfolding energy landscape , 2010, Proteomics.

[30]  Daniel J. Muller,et al.  One beta hairpin after the other: exploring mechanical unfolding pathways of the transmembrane beta-barrel protein OmpG. , 2009, Angewandte Chemie.

[31]  Luc Moulinier,et al.  Transmembrane Signaling across the Ligand-Gated FhuA Receptor Crystal Structures of Free and Ferrichrome-Bound States Reveal Allosteric Changes , 1998, Cell.

[32]  J. Kleinschmidt.  Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers. , 2006, Chemistry and physics of lipids.

[33]  T. Langer,et al.  Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines. , 2012, Biochimica et biophysica acta.

[34]  D. Müller,et al.  One β hairpin follows the other: exploring refolding pathways and kinetics of the transmembrane β-barrel protein OmpG. , 2011, Angewandte Chemie.