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Review
. 2014 Dec 15;564:265-80.
doi: 10.1016/j.abb.2014.02.011. Epub 2014 Mar 5.

Mechanistic Studies of the Biogenesis and Folding of Outer Membrane Proteins in Vitro and in Vivo: What Have We Learned to Date?

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Free PMC article
Review

Mechanistic Studies of the Biogenesis and Folding of Outer Membrane Proteins in Vitro and in Vivo: What Have We Learned to Date?

Lindsay M McMorran et al. Arch Biochem Biophys. .
Free PMC article

Abstract

Research into the mechanisms by which proteins fold into their native structures has been on-going since the work of Anfinsen in the 1960s. Since that time, the folding mechanisms of small, water-soluble proteins have been well characterised. By contrast, progress in understanding the biogenesis and folding mechanisms of integral membrane proteins has lagged significantly because of the need to create a membrane mimetic environment for folding studies in vitro and the difficulties in finding suitable conditions in which reversible folding can be achieved. Improved knowledge of the factors that promote membrane protein folding and disfavour aggregation now allows studies of folding into lipid bilayers in vitro to be performed. Consequently, mechanistic details and structural information about membrane protein folding are now emerging at an ever increasing pace. Using the panoply of methods developed for studies of the folding of water-soluble proteins. This review summarises current knowledge of the mechanisms of outer membrane protein biogenesis and folding into lipid bilayers in vivo and in vitro and discusses the experimental techniques utilised to gain this information. The emerging knowledge is beginning to allow comparisons to be made between the folding of membrane proteins with current understanding of the mechanisms of folding of water-soluble proteins.

Keywords: BAM complex; Outer membrane protein; Periplasmic chaperone; Protein folding; Protein stability; Φ-Value analysis.

Figures

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Fig. 1
Fig. 1
Schematic representation of funnel-shaped folding landscapes. Example of (a) a smooth folding landscape expected for a two-state folding mechanism where only the native and unfolded states are stably populated and (b) a rugged landscape wherein the polypeptide chain populates one or more intermediate structures which represent local energy minima. Reprinted from by permission from Macmillan Publishers Ltd: Nat. Struct. Mol. Biol. © 2009.
Fig. 2
Fig. 2
Example structures of integral membrane proteins. Structures of (a) the transmembrane segment of a glycophorin A monomer from human erythrocyte membranes solved by NMR spectroscopy (1AFO [228]); (b) bacteriorhodopsin, a seven-helical bundle from the purple membrane of Halobacterium salinarum (1C3 W [229]); (c) calcium ATPase 1 from the sarcoplasmic reticulum membrane of Oryctolagus cuniculus, a ten-helical bundle with a large cytoplasmic domain (1IWO [230]); (d) PagP, an 8-stranded palmitoyl transferase enzyme from E. coli (1THQ [206]); (e) the 8-stranded transmembrane domain of OmpA, an ion channel from E. coli (1BXW [231]), with the C-terminal periplasmic domain (structure currently not determined) represented by a red circle; (f) the 10-stranded OM protease, OmpT, from E. coli (1I78 [232]); (g) the 12-stranded, colicin-secreting phospholipase A, OmpLA, from E. coli (1QD5 [233]); (h) the OmpF porin, a trimer comprised of three 16-stranded β-barrels, from E. coli (2ZFG [234]) and (i) the 24-stranded translocation domain of PapC from E. coli (3FIP [235]). Unless otherwise specified, all structures were solved using X-ray crystallography. Proteins are coloured rainbow: violet (N-terminus) to red (C-terminus). In (h), a single OmpF monomer is coloured, while the remaining monomers are shown in greyscale. The approximate position of the membrane is indicated in all images with grey shading. All images were generated from the PDB files using the accession numbers given in brackets using UCSF Chimera molecular visualisation application .
Fig. 3
Fig. 3
The cell envelope of Gram-negative bacteria. (a) The cytoplasm of E. coli is surrounded by the inner membrane (IM), the periplasm and the outer membrane (OM). The IM is a symmetric phospholipid (shown in orange) bilayer containing integral α-helical membrane proteins. The OM is an asymmetric bilayer of phospholipid and lipopolysaccharide (LPS, shown in purple) and contains β-barrel integral membrane proteins. The periplasm is the aqueous compartment between the two membranes in which the peptidoglycan cell wall is found. Both membranes have associated lipoproteins on their periplasmic faces. (b) The lipid composition of the IM (light blue), inner leaflet of the OM (dark blue) and outer leaflet of the OM (white) in E. coli (percentages based on those reported in [64–66]). Structures of (c) LPS, (d) phosphatidylethanolamine, (e) phosphatidylglycerol and (f) cardiolipin are shown.
Fig. 4
Fig. 4
Schematic of the current model of biogenesis and chaperoning of OMPs in E. coli. (a) OMPs are synthesised on the ribosome before post-translational translocation across the inner membrane by the SecYEG translocon. Unfolded OMPs are then chaperoned across the periplasm to the β-barrel assembly machinery (BAM) complex, which aids folding and insertion into the OM. BAM complex proteins are labelled A–E, and the periplasmic polypeptide transport-associated (POTRA) domains of BamA are labelled P1-5. Horizontal black lines indicate the approximate position of the inner and outer membranes. (b) Flow diagram of the periplasmic and outer membrane-anchored proteins which may be implicated in OMP biogenesis. (a) was adapted from with permission from Elsevier, © 2013, while (b) was reproduced from with permission from John Wiley and Sons, © 2005.
Fig. 5
Fig. 5
Crystallographic structures of selected periplasmic chaperones. (a) Ribbon diagram of SurA coloured as follows N-terminal domain (blue), PPIase domain P1 (green), PPIase domain P2 (orange) and C-terminal domain (red) (1M5Y [80]). (b) Ribbon diagram of Skp trimer with the subunits A, B and C coloured in green, magenta and blue, respectively, (1U2M [104]). The tips of the α-helices in subunits A and B have been modelled. (c) Ribbon diagram of the FkpA dimer showing the N-terminal chaperone domains (red and orange) through which dimerisation occurs and the C-terminal PPIase domains (blue) (1Q6H [109]). (d) Ribbon diagram of the Spy dimer with the monomers coloured individually in red and blue (3O39 [114]). (a), (c) and (d) were generated from PDB files using the accession numbers given in brackets using UCSF Chimera molecular visualisation application . (b) was reproduced from with permission from Elsevier, © 2004.
Fig. 6
Fig. 6
Structure of the BAM complex. (a) Schematic of the E. coli BAM complex with BAM proteins labelled A–E and POTRA domains labelled P1–5 (reproduced from with permission from Elsevier, © 2013). (b) Crystal structure of N. gonorrhoeae BamA (4K3B [120]). The β-barrel domain is shown in orange. POTRA domains are labelled as in (a) and are shown in pink, blue, green, purple and yellow. (c) Crystal structure of E. coli BamB (3P1L [130]). The blades of the β-propeller structure are coloured individually and labelled 1–8. (d) Crystal structure of the N-terminal domain of E. coli BamC (dark blue) bound to BamD (2YHC [125]). The five TPR motifs of BamD coloured in light blue, yellow, green, pink and orange. (e) Lowest energy structure of E. coli BamE solved by NMR spectroscopy (2KXX [237]). All images were generated from the PDB files using the accession numbers given in brackets using UCSF Chimera molecular visualisation application .
Fig. 7
Fig. 7
Schematic of the principles of Φ-value analysis. (a) A mutation (shown as a red dot) is made in a region of the protein which is native-like in the transition state (‡) leading to equal destabilisation of ‡ and the native state (N) resulting in a Φ-value of 1 or (b) a mutation is made in a region of the protein which is unfolded in ‡ but structured in the native state, leading to destabilisation of N only and a Φ-value of 0. It is assumed that the mutation does not affect the free energy of the unfolded ensemble (U). Abbreviations: ΔΔG°U–N refers to the difference in the free energy of folding upon mutation and ΔΔG°U–‡ refers to the difference in the free energy between U and ‡ upon mutation. This image was adapted with permission from G.H.M. Huysmans.
Fig. 8
Fig. 8
Proposed mechanism of folding and insertion of the OmpA β-barrel domain into lipid bilayers in vitro. (a) Depicts an unfolded, membrane-bound state, (b) depicts a partially folded and inserted state, and (c) depicts the native state. Coloured circles indicate the location of tryptophan residues in the OmpA structure. Reproduced from with permission from Elsevier, © 2011.
Fig. 9
Fig. 9
Φ-Value analysis of HT PagP. ΦF-values determined from kinetic analysis of HT PagP variants are mapped onto a ribbon diagram (left) and a topology model (right). Regions with ΦF-values close to 0 are shown in red, regions with ΦF-values close to 1 are shown in blue, intermediate ΦF-values are shown in purple, ΦF-values less than 1 are shown in orange and undetermined ΦF-values are grey. Reproduced with permission from .
Fig. 10
Fig. 10
Schematic of the effects of Skp and SurA on the refolding of PagP. SurA and PagP do not interact stably under the conditions of the refolding assay. Skp readily interacts with PagP, retarding the PagP folding rate into zwitterionic liposomes, but accelerating the folding rate of PagP into negatively charged liposomes in a manner dependent on the ionic strength of the buffer. Additionally, the holdase activity of Skp was demonstrated by its ability to rescue the folding and membrane insertion of HT PagP under conditions which strongly favour aggregation of this construct. This figure was adapted from with permission from Elsevier, © 2013.

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