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