Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Impact of holdase chaperones Skp and SurA on the folding of β-barrel outer-membrane proteins

Nature Structural & Molecular Biology volume 22, pages 795–802 (2015)Cite this article

Subjects

Abstract

Chaperones increase the folding yields of soluble proteins by suppressing misfolding and aggregation, but how they modulate the folding of integral membrane proteins is not well understood. Here we use single-molecule force spectroscopy and NMR spectroscopy to observe the periplasmic holdase chaperones SurA and Skp shaping the folding trajectory of the large β-barrel outer-membrane receptor FhuA from Escherichia coli. Either chaperone prevents FhuA from misfolding by stabilizing a dynamic, unfolded state, thus allowing the substrate to search for structural intermediates. During this search, the SurA-chaperoned FhuA polypeptide inserts β-hairpins into the membrane in a stepwise manner until the β-barrel is folded. The membrane acts as a free-energy sink for β-hairpin insertion and physically separates transient folds from chaperones. This stabilization of dynamic unfolded states and the trapping of folding intermediates funnel the FhuA polypeptide toward the native conformation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mechanical unfolding steps of FhuA, as detected by SMFS.
Figure 2: Experimental assay to characterize the chaperone-modulated refolding of individual FhuA receptors.
Figure 3: The periplasmic chaperones Skp and SurA prevent the FhuA substrate from misfolding, and SurA supports the insertion and folding of individual β-hairpins.
Figure 4: The action of the periplasmic chaperones Skp and SurA is independent of the length of the unfolded FhuA polypeptide.
Figure 5: Binding affinity, ensemble state and lifetime of chaperone-bound FhuA substrates.
Figure 6: Refolding kinetics and mechanical stability of FhuA substrates refolded in the presence and absence of SurA.
Figure 7: Folding pathways and free-energy landscape of FhuA receptors.

Similar content being viewed by others

References

  1. Engel, A. & Gaub, H.E. Structure and mechanics of membrane proteins. Annu. Rev. Biochem. 77, 127–148 (2008).

    Article  CAS  Google Scholar 

  2. Zoldák, G. & Rief, M. Force as a single molecule probe of multidimensional protein energy landscapes. Curr. Opin. Struct. Biol. 23, 48–57 (2013).

    Article  Google Scholar 

  3. Kedrov, A., Ziegler, C., Janovjak, H., Kühlbrandt, W. & Müller, D.J. Controlled unfolding and refolding of a single sodium-proton antiporter using atomic force microscopy. J. Mol. Biol. 340, 1143–1152 (2004).

    Article  CAS  Google Scholar 

  4. Kessler, M., Gottschalk, K.E., Janovjak, H., Muller, D.J. & Gaub, H.E. Bacteriorhodopsin folds into the membrane against an external force. J. Mol. Biol. 357, 644–654 (2006).

    Article  CAS  Google Scholar 

  5. Damaghi, M., Koster, S., Bippes, C.A., Yildiz, O. & Müller, D.J. One β-hairpin follows the other: exploring refolding pathways and kinetics of the transmembrane β-barrel protein OmpG. Angew. Chem. Int. Edn. Engl. 50, 7422–7424 (2011).

    Article  CAS  Google Scholar 

  6. Bukau, B., Weissman, J. & Horwich, A. Molecular chaperones and protein quality control. Cell 125, 443–451 (2006).

    Article  CAS  Google Scholar 

  7. Hartl, F.U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

    Article  CAS  Google Scholar 

  8. Bechtluft, P. et al. Direct observation of chaperone-induced changes in a protein folding pathway. Science 318, 1458–1461 (2007).

    Article  CAS  Google Scholar 

  9. Hillger, F. et al. Probing protein-chaperone interactions with single-molecule fluorescence spectroscopy. Angew. Chem. Int. Edn. Engl. 47, 6184–6188 (2008).

    Article  CAS  Google Scholar 

  10. Sharma, S. et al. Monitoring protein conformation along the pathway of chaperonin-assisted folding. Cell 133, 142–153 (2008).

    Article  CAS  Google Scholar 

  11. Kaiser, C.M. et al. Tracking UNC-45 chaperone-myosin interaction with a titin mechanical reporter. Biophys. J. 102, 2212–2219 (2012).

    Article  CAS  Google Scholar 

  12. Mashaghi, A. et al. Reshaping of the conformational search of a protein by the chaperone trigger factor. Nature 500, 98–101 (2013).

    Article  CAS  Google Scholar 

  13. Mashaghi, A., Kramer, G., Lamb, D.C., Mayer, M.P. & Tans, S.J. Chaperone action at the single-molecule level. Chem. Rev. 114, 660–676 (2014).

    Article  CAS  Google Scholar 

  14. Nunes, J.M., Mayer-Hartl, M., Hartl, F.U. & Müller, D.J. Action of the Hsp70 chaperone system observed with single proteins. Nat. Commun. 6, 6307 (2015).

    Article  CAS  Google Scholar 

  15. Engelman, D.M. et al. Membrane protein folding: beyond the two stage model. FEBS Lett. 555, 122–125 (2003).

    Article  CAS  Google Scholar 

  16. Bowie, J.U. Solving the membrane protein folding problem. Nature 438, 581–589 (2005).

    Article  CAS  Google Scholar 

  17. Cymer, F., von Heijne, G. & White, S.H. Mechanisms of integral membrane protein insertion and folding. J. Mol. Biol. 427, 999–1022 (2015).

    Article  CAS  Google Scholar 

  18. Ruiz, N., Kahne, D. & Silhavy, T.J. Advances in understanding bacterial outer-membrane biogenesis. Nat. Rev. Microbiol. 4, 57–66 (2006).

    Article  Google Scholar 

  19. Bos, M.P., Robert, V. & Tommassen, J. Biogenesis of the gram-negative bacterial outer membrane. Annu. Rev. Microbiol. 61, 191–214 (2007).

    Article  CAS  Google Scholar 

  20. Knowles, T.J., Scott-Tucker, A., Overduin, M. & Henderson, I.R. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly. Nat. Rev. Microbiol. 7, 206–214 (2009).

    Article  CAS  Google Scholar 

  21. Ferguson, A.D., Hofmann, E., Coulton, J.W., Diederichs, K. & Welte, W. Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. Science 282, 2215–2220 (1998).

    Article  CAS  Google Scholar 

  22. Locher, K.P. et al. Transmembrane signaling across the ligand-gated FhuA receptor: crystal structures of free and ferrichrome-bound states reveal allosteric changes. Cell 95, 771–778 (1998).

    Article  CAS  Google Scholar 

  23. Thoma, J., Bosshart, P., Pfreundschuh, M. & Müller, D.J. Out but not in: the large transmembrane β-barrel protein FhuA unfolds but cannot refold via β-hairpins. Structure 20, 2185–2190 (2012).

    Article  CAS  Google Scholar 

  24. Jarchow, S., Lück, C., Görg, A. & Skerra, A. Identification of potential substrate proteins for the periplasmic Escherichia coli chaperone Skp. Proteomics 8, 4987–4994 (2008).

    Article  CAS  Google Scholar 

  25. Vertommen, D., Ruiz, N., Leverrier, P., Silhavy, T.J. & Collet, J.F. Characterization of the role of the Escherichia coli periplasmic chaperone SurA using differential proteomics. Proteomics 9, 2432–2443 (2009).

    Article  CAS  Google Scholar 

  26. Denoncin, K., Schwalm, J., Vertommen, D., Silhavy, T.J. & Collet, J.F. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics. Proteomics 12, 1391–1401 (2012).

    Article  CAS  Google Scholar 

  27. Sklar, J.G., Wu, T., Kahne, D. & Silhavy, T.J. Defining the roles of the periplasmic chaperones SurA, Skp, and DegP in Escherichia coli. Genes Dev. 21, 2473–2484 (2007).

    Article  CAS  Google Scholar 

  28. Hennecke, G., Nolte, J., Volkmer-Engert, R., Schneider-Mergener, J. & Behrens, S. The periplasmic chaperone SurA exploits two features characteristic of integral outer membrane proteins for selective substrate recognition. J. Biol. Chem. 280, 23540–23548 (2005).

    Article  CAS  Google Scholar 

  29. Qu, J., Mayer, C., Behrens, S., Holst, O. & Kleinschmidt, J.H. The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions. J. Mol. Biol. 374, 91–105 (2007).

    Article  CAS  Google Scholar 

  30. Bulieris, P.V., Behrens, S., Holst, O. & Kleinschmidt, J.H. Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide. J. Biol. Chem. 278, 9092–9099 (2003).

    Article  CAS  Google Scholar 

  31. Patel, G.J., Behrens-Kneip, S., Holst, O. & Kleinschmidt, J.H. The periplasmic chaperone Skp facilitates targeting, insertion, and folding of OmpA into lipid membranes with a negative membrane surface potential. Biochemistry 48, 10235–10245 (2009).

    Article  CAS  Google Scholar 

  32. McMorran, L.M., Bartlett, A.I., Huysmans, G.H., Radford, S.E. & Brockwell, D.J. Dissecting the effects of periplasmic chaperones on the in vitro folding of the outer membrane protein PagP. J. Mol. Biol. 425, 3178–3191 (2013).

    Article  CAS  Google Scholar 

  33. Burmann, B.M., Wang, C. & Hiller, S. Conformation and dynamics of the periplasmic membrane-protein–chaperone complexes OmpX–Skp and tOmpA–Skp. Nat. Struct. Mol. Biol. 20, 1265–1272 (2013).

    Article  CAS  Google Scholar 

  34. Müller, D.J. & Engel, A. Atomic force microscopy and spectroscopy of native membrane proteins. Nat. Protoc. 2, 2191–2197 (2007).

    Article  Google Scholar 

  35. Bustamante, C., Marko, J.F., Siggia, E.D. & Smith, S. Entropic elasticity of lambda-phage DNA. Science 265, 1599–1600 (1994).

    Article  CAS  Google Scholar 

  36. Kedrov, A., Janovjak, H., Sapra, K.T. & Müller, D.J. Deciphering molecular interactions of native membrane proteins by single-molecule force spectroscopy. Annu. Rev. Biophys. Biomol. Struct. 36, 233–260 (2007).

    Article  CAS  Google Scholar 

  37. Sapra, K.T. et al. One beta hairpin after the other: exploring mechanical unfolding pathways of the transmembrane beta-barrel protein OmpG. Angew. Chem. Int. Edn. Engl. 48, 8306–8308 (2009).

    Article  CAS  Google Scholar 

  38. Bosshart, P.D. et al. The transmembrane protein KpOmpA anchoring the outer membrane of Klebsiella pneumoniae unfolds and refolds in response to tensile load. Structure 20, 121–127 (2012).

    Article  CAS  Google Scholar 

  39. Hensen, U. & Müller, D.J. Mechanistic explanation of different unfolding behaviors observed for transmembrane and soluble β-barrel proteins. Structure 21, 1317–1324 (2013).

    Article  CAS  Google Scholar 

  40. Kedrov, A., Krieg, M., Ziegler, C., Kuhlbrandt, W. & Muller, D.J. Locating ligand binding and activation of a single antiporter. EMBO Rep. 6, 668–674 (2005).

    Article  CAS  Google Scholar 

  41. Damaghi, M. et al. pH-dependent interactions guide the folding and gate the transmembrane pore of the beta-barrel membrane protein OmpG. J. Mol. Biol. 397, 878–882 (2010).

    Article  CAS  Google Scholar 

  42. Zocher, M., Zhang, C., Rasmussen, S.G., Kobilka, B.K. & Müller, D.J. Cholesterol increases kinetic, energetic, and mechanical stability of the human beta2-adrenergic receptor. Proc. Natl. Acad. Sci. USA 109, E3463–E3472 (2012).

    Article  CAS  Google Scholar 

  43. Bippes, C.A. et al. Peptide transporter DtpA has two alternate conformations, one of which is promoted by inhibitor binding. Proc. Natl. Acad. Sci. USA 110, E3978–E3986 (2013).

    Article  CAS  Google Scholar 

  44. Serdiuk, T., Sugihara, J., Mari, S.A., Kaback, H.R. & Müller, D.J. Observing a lipid-dependent alteration in single lactose permeases. Structure 23, 754–761 (2015).

    Article  CAS  Google Scholar 

  45. Doyle, S.M., Genest, O. & Wickner, S. Protein rescue from aggregates by powerful molecular chaperone machines. Nat. Rev. Mol. Cell Biol. 14, 617–629 (2013).

    Article  CAS  Google Scholar 

  46. Moon, C.P., Zaccai, N.R., Fleming, P.J., Gessmann, D. & Fleming, K.G. Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm. Proc. Natl. Acad. Sci. USA 110, 4285–4290 (2013).

    Article  CAS  Google Scholar 

  47. Gruss, F. et al. The structural basis of autotransporter translocation by TamA. Nat. Struct. Mol. Biol. 20, 1318–1320 (2013).

    Article  CAS  Google Scholar 

  48. Noinaj, N. et al. Structural insight into the biogenesis of β-barrel membrane proteins. Nature 501, 385–390 (2013).

    Article  CAS  Google Scholar 

  49. Noinaj, N., Kuszak, A.J., Balusek, C., Gumbart, J.C. & Buchanan, S.K. Lateral opening and exit pore formation are required for BamA function. Structure 22, 1055–1062 (2014).

    Article  CAS  Google Scholar 

  50. Kim, K.H., Aulakh, S. & Paetzel, M. The bacterial outer membrane beta-barrel assembly machinery. Protein Sci. 21, 751–768 (2012).

    Article  CAS  Google Scholar 

  51. Stroud, D.A., Meisinger, C., Pfanner, N. & Wiedemann, N. Assembling the outer membrane. Science 328, 831–832 (2010).

    Article  CAS  Google Scholar 

  52. Hagan, C.L., Silhavy, T.J. & Kahne, D. β-barrel membrane protein assembly by the Bam complex. Annu. Rev. Biochem. 80, 189–210 (2011).

    Article  CAS  Google Scholar 

  53. Prilipov, A., Phale, P.S., Van Gelder, P., Rosenbusch, J.P. & Koebnik, R. Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. FEMS Microbiol. Lett. 163, 65–72 (1998).

    Article  CAS  Google Scholar 

  54. Oesterhelt, F. et al. Unfolding pathways of individual bacteriorhodopsins. Science 288, 143–146 (2000).

    Article  CAS  Google Scholar 

  55. Efron, B. & Tibshirani, R.J. An introduction to the Bootstrap (Chapman and Hall/CRC Press, New York, 1993).

  56. Marko, J.F. & Siggia, E.D. Stretching DNA. Macromolecules 28, 8759–8770 (1995).

    Article  CAS  Google Scholar 

  57. Odijk, T. Stiff chains and filaments under tension. Macromolecules 28, 7016–7018 (1995).

    Article  CAS  Google Scholar 

  58. Lawrence, J.J., Berne, L., Ouvrier-Buffet, J.L. & Piette, L.H. Spin-label study of histone H1-DNA interaction: comparative properties of the central part of the molecule and the N and C-amino tails. Eur. J. Biochem. 107, 263–269 (1980).

    Article  CAS  Google Scholar 

  59. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc. Natl. Acad. Sci. USA 94, 12366–12371 (1997).

    Article  CAS  Google Scholar 

  60. Zhu, G., Xia, Y., Nicholson, L.K. & Sze, K.H. Protein dynamics measurements by TROSY-based NMR experiments. J. Magn. Reson. 143, 423–426 (2000).

    Article  CAS  Google Scholar 

  61. Lee, D., Hilty, C., Wider, G. & Wüthrich, K. Effective rotational correlation times of proteins from NMR relaxation interference. J. Magn. Reson. 178, 72–76 (2006).

    Article  CAS  Google Scholar 

  62. Güntert, P., Dötsch, V., Wider, G. & Wüthrich, K. Processing of multi-dimensional NMR data with the new software PROSA. J. Biomol. NMR 2, 619–629 (1992).

    Article  Google Scholar 

  63. Keller, R.L.J. The Computer Aided Resonance Assignment Tutorial (Cantina Verlag, Goldau, Switzerland, 2004).

Download references

Acknowledgements

We thank R. Newton for critical discussion. This work was supported by the Swiss National Science Foundation (grants PP00P3_128419 to S.H. and 200021_134521 to D.J.M.), the National Center of Competence in Research ('NCCR Molecular Systems Engineering' to D.J.M.) and the European Research Council (FP7 contract MOMP 281764 to S.H.).

Author information

Author notes

  1. Johannes Thoma and Björn M Burmann: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule Zürich, Basel, Switzerland

    Johannes Thoma & Daniel J Müller

  2. Biozentrum, University of Basel, Basel, Switzerland

    Björn M Burmann & Sebastian Hiller

Authors
  1. Johannes Thoma
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Björn M Burmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sebastian Hiller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daniel J Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.T. cloned, expressed, purified and reconstituted FhuA and performed the SMFS experiments. B.M.B. expressed and purified SurA and Skp and performed the NMR experiments. All authors designed experiments, analyzed experimental data and wrote the paper.

Corresponding authors

Correspondence to Sebastian Hiller or Daniel J Müller.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Purification, folding and reconstitution of FhuA receptors into E. coli lipid membranes.

(a) SDS-PAGE of purified FhuA solubilized in the presence of N,N-dimethyldodecylamine N-oxide (LDAO). Lane 1 shows the FhuA sample before thermal denaturation. Folded FhuA migrates in a major band around ≍55 kDa with a smeared pattern1. Lane 2 shows the FhuA sample after heating to 95°C migrating in one band around ≍75 kDa. (b) Circular dichroism (CD) spectrum of FhuA. The spectrum shows a pattern distinct for folded β-barrel proteins in agreement with earlier experiments2. (c) AFM topograph of an FhuA proteoliposome adsorbed to freshly cleaved mica. Upon attachment to mica the proteoliposomes open and adsorb as single layered membrane patches. The lipid membrane shows sparsely distributed and densely packed areas of FhuA. (d) Higher magnification of the area dashed in c, featuring regions with sparsely distributed (SD), densely packed (DP), and two-dimensional (2D) crystalline assemblies of FhuA. Refolding experiments were selectively performed on densely packed regions. AFM topographs were recorded in buffer solution (25 mM MES, 150 mM NaCl, pH 6.5) at room temperature (≍22°C).

1. Locher, K.P. & Rosenbusch, J.P. Oligomeric states and siderophore binding of the ligand-gated FhuA protein that forms channels across Escherichia coli outer membranes. Eur. J. Biochem. 247, 770-775 (1997).

2. Moeck, G.S. et al. Ligand-induced conformational change in the ferrichrome-iron receptor of Escherichia coli K-12. Mol. Microbiol. 22, 459-471 (1996).

Supplementary Figure 2 The AFM tip preferentially attaches to the N-terminal domain of FhuA, from which mechanical unfolding proceeds.

SDS-PAGE of full-length FhuA and FhuAΔP lacking the N-terminal plug domain. (b) Secondary structure models of FhuA and FhuAΔP. Labeled are the N-terminal plug domain (P) and the 11 β-hairpins H1–H11 forming the transmembrane β-barrel. Stars highlight the contour lengths of the FhuA polypeptide, which is given in brackets in amino acids (aa). (c) Superimposition of 80 force-distance curves obtained from unfolding full-length FhuA from E. coli lipid bilayers. Worm-like-chain (WLC) curves were fitted to individual force peaks (grey solid and dashed curves) to reveal the contour length of the unfolded polypeptide stretched in each unfolding step. At the end of each WLC fit the contour length is given in aa. (d) Superimposition of 53 force-distance curves obtained from unfolding FhuAΔP from E. coli lipid bilayers. The superimposition shows the same characteristic pattern of force peaks as observed for full-length FhuA in c except that all peak positions shifted towards shorter distances by 138 ± 4 aa (≍45 nm). The residual N-terminal domain in FhuAΔP comprises 20 residues, as compared to 161 aa of full-length FhuA. The observed shift of 138 aa corresponds to the expected difference of 141 aa and directly confirms that both FhuA and FhuAΔP are mechanically pulled from the N-terminus. Thus, the AFM tip pressed onto FhuA preferentially attaches, non-specifically to the N-terminal domain. The alternative scenario that FhuA and FhuAΔP would be pulled from the C-terminus would not shift the force peak pattern for FhuAΔP and can thus be ruled out3. Furthermore, the observed preference for the non-specific attachment of the AFM tip to the N-terminal domain is in full agreement with the relative sizes of these two domains (161 aa vs. 2 aa for full-length FhuA).

3. Thoma, J., Bosshart, P., Pfreundschuh, M. & Muller, D.J. Out but not in: the large transmembrane β-barrel protein FhuA unfolds but cannot refold via β-hairpins. Structure 20, 2185-90 (2012).

Supplementary Figure 3 β-hairpins of the partially unfolded β-barrel of FhuA remain stably embedded in the lipid membrane and maintain their native fold.

Shown are force-distance curves recorded upon mechanically unfolding of single FhuA receptors. Initially, the FhuA β-barrel was partially unfolded (red force-distance curves) until (a) β-hairpin H5, corresponding to a pulling distance of ≍130 nm, (b) β-hairpin H6, corresponding to a pulling distance of ≍155 nm, or (c) β-hairpin H8, corresponding to a pulling distance of ≍180 nm. These different degrees of unfolding intermediates left either (a) 6 β-hairpins, (b) 5 β-hairpins, or (c) 3 β-hairpins of the FhuA β-barrel folded in the membrane. Subsequently, the unfolded polypeptide was approached close to the membrane (≍10 nm), where it was kept for 1 s. After this time passed, the receptor was completely unfolded while recording the blue force-distance curve. As reference, the WLC curves (grey solid and dashed curves) obtained from fitting the native fingerprint pattern of FhuA are indicated (see Fig. 1b,c). The force peaks recorded upon unfolding of the residual β-hairpins of partially unfolded FhuA are at the positions observed for the native FhuA receptor. Thus, these residual β-hairpins do not undergo major conformational changes during the 1 s left for reassembly. Experiments were conducted of FhuA reconstituted in E. coli lipid membranes, in buffer solution (25 mM MES, 150 mM NaCl, pH 6.5), at room temperature, and in the absence of chaperones.

Supplementary Figure 4 Criteria for classifying the refolded FhuA substrate.

(a) Pair of consecutive force-distance curves of FhuA unfolding. The red force-distance curve records the partial unfolding of a single natively folded FhuA. Each force peak describes an unfolding step of FhuA (Fig. 1). To correlate these unfolding steps to the fingerprint spectrum of native FhuA we superimposed the WLC curves (grey solid and dashed curves), which were obtained from fitting the average fingerprint spectrum of native FhuA (comp. Fig. 1b,c). Above each WLC curve the contour length of the unfolded and stretched polypeptide segment (number of amino acids given in brackets) and the name of the unfolded structural segment (i.e., the N-terminal plug domain P or β-hairpins H1–H11) are given. This superimposition of a single force-distance curve and the WLC curves highlights the agreement between the unfolding steps of a single native FhuA and the unfolding steps of the ensemble average. The partial unfolding curve of native FhuA shows an example in which β-hairpin H3 shows a double force peak. This second unfolding force peak following the first unfolding peak indicates that the β-hairpin unfolded via an intermediate. Although such an unfolding intermediate of a β-hairpin can be observed, it is never observed to occur alone in the absence of the first unfolding fore peak. After partial unfolding, the AFM tip was brought in close proximity to the membrane (≍10 nm) to allow the unfolded polypeptide refolding for 1 s. Then, the partially unfolded FhuA was completely unfolded (blue force-distance curve). (b) Enlarged region highlighted in (a). Above the black WLC curve the average contour length and the full-width half maximum (FWHM) from WLC curves fitting the native fingerprint spectrum are given (Fig. 1c). The gray shaded area embedding the WLC curve gives the FWHM of the contour length average. The orange WLC curve, which was fitted to the blue refolding force peak, lies outside the average contour length ± FHWM of the native force peak. Therefore, this refolding step constitutes a non-native structural element and the refolding event was classified as ‘misfolded’. (c) Pair of consecutive force-distance curves one recorded upon partially unfolding a single native FhuA (red curve) and one recorded of the same FhuA molecule after a refolding time of 1 s (blue curve). (d) Enlarged region highlighted in (c). The two light blue WLC curves fitted to the two refolding force peaks lay inside of the average contour lengths ± FWHM of the native fingerprint spectrum. Therefore, this refolding FhuA polypeptide shows two correctly folded β-hairpins and was thus classified as ‘folded β-hairpins’. In the absence of any refolding force peaks (examples shown in Fig. 2 or Supplementary Fig. 5), a FhuA substrate would be classified as ‘unfolded’.

Supplementary Figure 5 Representative force-distance curves of FhuA refolded in different conditions.

Primary unfolding force-distance curves recorded of native FhuA are shown in red, secondary unfolding curves of refolded FhuA in blue. FhuA was refolded in (a) buffer, (b) the presence of 1 μM Skp, (c) the presence of 1 μM SurA, (d) the presence of 1 μM Skp and 1 μM SurA, (e) the presence of 1 μM BSA, and (f) the presence of 1 μM lysozyme. The classification of each force-distance curve into the categories "unfolded" (U), "misfolded" (M), and "folded β-hairpins" (F) is indicated. The average WLC fits (grey solid and dashed curves) of the native fingerprint spectrum of FhuA (see Fig. 1b,c) were used to evaluate the positions of the refolding force peaks and thus to classify the individual refolding events (Supplementary Fig. 4). Black arrows indicate force peaks corresponding to correctly folded secondary structure elements. White arrows mark force peaks indicating misfolding intermediates. The refolding time was 1 s. Experiments were conducted of FhuA reconstituted in E. coli lipid membranes, in buffer solution (25 mM MES, 150 mM NaCl, pH 6.5) and at room temperature.

Supplementary Figure 6 Biochemical characterization of chaperones and chaperone–FhuA complexes.

(a) Purity of protein samples, as assessed by SDS-PAGE. (b) SDS-PAGE of the peak fraction of the size exclusion chromatography runs depicted in panel (c), as indicated by letters A-F. (c) Elution profiles of membrane proteins, chaperones, and their complexes, from size-exclusion chromatography. Profiles were recorded at 8°C in 25 mM Hepes, 50 mM NaCl, 1% Glycerol, pH 7.5 on a Superdex200 increase size exclusion column (GE Healthcare). Protein concentrations applied were: 20 µM Skp trimer; 5 µM Skp-FhuA; 10 µM SurA; 5 µM SurA-FhuA; 0.5 µM FhuA. The column void volume and the molecular weights of a standard calibration curve are indicated. Note that in the absence of chaperones the transmembrane protein FhuA is not soluble and thus does not elute as a peak. Furthermore, the peak maximum of free SurA in FhuA-SurA samples (peak F) is shifted relative to apo SurA (peak D). This may result from dynamic binding events in the ensemble, shifting the elution position towards larger effective molecular weights. (d) Pulldown experiments of FhuA and chaperones. Each experiment comprises a wash (W) and an elution (E) fraction from a Ni2+-affinity column. Above the SDS-PAGE gel, the combinations of proteins that were incubated and then loaded on the column are indicated. Lane 11 contains the molecular weight marker (M). The expected protein positions are indicated on the left hand side.

Supplementary Figure 7 SDS-PAGE analysis of chaperone–substrate complexes.

(a) Reference bands for 20 µM of SurA and 20 µM FhuA along with SDS-PAGE analysis of total stoichiometries in SurA-FhuA complexes as a function of biochemical condition and time. (b) Same for the Skp trimer. Impurities are marked with an asterisk. (c) Concentration of FhuA in FhuA-SurA samples, as determined by quantification of SDS-PAGE intensities using the software ImageJ. (d) Same for the Skp trimer. For SurA-FhuA and Skp-FhuA complexes, the highest stoichiometry for FhuA was observed at pH 6.5 for low salt condition. Under all experimental conditions, SurA is at least 3-fold higher concentrated than FhuA, which is in agreement with the size exclusion chromatography analysis (Supplementary Fig. 6), resulting in migration corresponding to a 2:1 SurA:FhuA ratio, in the presence of a significant amount of free SurA. Under all experimental conditions, the Skp trimer was at least 2-fold higher concentrated than FhuA, which is in agreement with the size exclusion chromatography analysis. Overall, for both SurA and the Skp trimer, the data is compatible with chaperone:FhuA stoichiometries of 2:1 and above.

Supplementary Figure 8 In the presence of SurA, the unfolded FhuA receptor can fully refold.

Shown are representative force-distance curves recorded of the FhuA substrate, which has been refolded for 2 s, 5 s and 10 s. Force-distance curves recorded upon initially unfolding the native FhuA receptor are shown in red and force-distance curves recorded of the refolded FhuA substrate are shown in blue. FhuA receptors were unfolded and refolded as described (Fig. 2) in the presence of 1 μM SurA. Depending on the refolding time of 2 s (a), 5 s (b) and 10 s (c) the force-distance curves show different amounts of correctly folded β-hairpins indicating a partially or fully refolded β-barrel. The average WLC fits (grey solid and dashed curves) of the native fingerprint spectrum of FhuA (Fig. 1b,c) allow to evaluate whether the positions of the force peaks observed after refolding correspond to those observed for the native FhuA receptor.

Supplementary Figure 9 Comparison of two different WLC models fitting the force-distance curve recorded upon unfolding of native FhuA.

(a) Simple (black curves) and modified, extensible (blue curves) WLC model fitted to a force-distance curve (red curve) recorded upon mechanically unfolding a single FhuA receptor. Each unfolding force peak was fitted either with a simple WLC model (i.e., the Marko-Siggia model4 using a persistence length of 0.4 nm), or with an extensible WLC model (i.e., the Odijk model5 using a persistence length of 0.4 nm and an elastic modulus K0 with: 500 pN < K0 < 5000 pN). Fits were performed using the Levenberg-Marquardt least-squares method6,7. (b) The residuals quantifying the differences between WLC fits and experimental unfolding force peak shown in (a). The residuals show that for most force peaks both WLC models yield very similar fits with only minor differences. Thus, the extensible WLC does not describe the SMFS data better. Residuals from the simple WLC fits are in black, residuals from the extensible WLC fits are in blue. (c) Root mean square (RMS) of the residuals of WLC fits obtained from fitting eight different force-distance curves. RMS values corresponding to simple WLC fits are in black, RMS values corresponding to extensible WLC fits are in blue. Because the RMS of the residuals is similar we can conclude that the extensible WLC model does not significantly improve the fit quality compared to the simple WLC model.

The motivation of this supplementary figure was to test whether we can further improve our WLC fits so that they more precisely follow the shape of the unfolding force peaks. Therefore we compared fits of unfolding force peaks either using a simple WLC model, or an extensible WLC model. Because the extensible WLC model did not significantly improve the fitting we decided to stay with the simple WLC model to determine the relative position (or contour length) of force peaks. Furthermore, we note that the outcome of the analysis of unfolded, misfolded and folding events is largely independent of the type of WLC model. This independency is a result of the fact that in our analysis the WLC fits are used to compare the positions (or contour lengths) of the force peaks detected upon unfolding of native FhuA with the unfolding force peaks detected for refolded FhuA (see Supplementary Fig. 4).

4. Marko, J.F. & Siggia, E.D. Stretching DNA. Macromolecules 28, 8759 (1995).

5. Odijk, T. Stiff chains and filaments under tension. Macromolecules 28, 7016-7018 (1995).

6. Levenberg, K. A method for the solution of certain non-linear problems in least squares. Q. J. Appl. Math. II, 164-168 (1944).

7. Marquardt, D. An algorithm for least-squares estimation of nonlinear parameters. J. Soc. Industr. Appl. Math. 11, 431-441 (1963).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 1–3 (PDF 2663 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Thoma, J., Burmann, B., Hiller, S. et al. Impact of holdase chaperones Skp and SurA on the folding of β-barrel outer-membrane proteins. Nat Struct Mol Biol 22, 795–802 (2015). https://doi.org/10.1038/nsmb.3087

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3087

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing