Monitoring Membrane Protein Folding Assisted by Insertases and Translocases Using AFM-Based Single-Molecule Force Spectroscopy

Figures

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  1. Transport, folding and insertion pathways of inner and outer membrane proteins in Gram-negative bacteria. Nascent polypeptides are sorted at the ribosome into co- or post-translational pathways. In the cotranslational pathway, signal recognition particle (SRP) and FtsY target ribosome–nascent chain complexes to the SecYEG translocon. In the post-translational pathway, cytosolic chaperones (trigger factor (TF), SecB) maintain substrates in an unfolded state for SecA-dependent delivery to SecYEG. SecYEG mediates membrane insertion of inner membrane proteins (IMPs), assisted by YidC and the SecDF–YajC complex, or translocation of outer membrane protein precursors into the periplasm following signal peptide cleavage by SPase I. In the periplasm, SurA transports substrates to the β-barrel assembly machinery (BAM) complex for folding and insertion into the outer membrane, while Skp and DegP contribute to quality control. Adapted with permission from ref. Copyright 2022 Elsevier.

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  2. Mechanical unfolding fingerprint of native LacY. (a) Schematic of mechanically unfolding and extracting a native LacY from a phospholipid membrane (POPE:POPG) using AFM-based SMFS. The tip of the AFM cantilever is pressed onto LacY (PDB: 1PV7), enabling nonspecific attachment at the extended C-terminus (polyGly-LacY). Retraction of the cantilever applies a mechanical force that unfolds LacY in a stepwise manner until being fully extracted from the membrane. (b) Force–distance curve from the unfolding of a single LacY. Raw data (pale red) and a smoothed trace (Savitzky-Golay filter, dark red) are shown. Each force peak is fitted with the worm-like chain (WLC) model , (gray curves) to calculate contour length of the unfolded polypeptide segment (in amino acids on the top of each WLC curve). (c) Density plot of 280 superimposed force–distance curves, each recording the mechanical unfolding of a single LacY. (d) Structural segments S1–S10 of LacY that are detected upon mechanical unfolding and mapped to the LacY secondary structure. Transmembrane α-helices are labeled I–XII. Reproduced with permission from ref. Copyright 2019 The American Association for the Advancement of Science.

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  3. SMFS detects two classes of unfolding patterns of LacY in the POPG membrane. (a, b) Density plots of superimposed force–distance curves, which were recorded upon the mechanical extraction and unfolding of LacY from POPG membranes. The plots show two classes (1 and 2) of force peak patterns. Colored WLC fits indicate mean contour lengths of the individual force peaks. n indicates the number of force–distance curves analyzed. (c, d) Contour length histograms for class 1 (c) and class 2 (d) force peak patterns, fitted using a Gaussian mixture model. Ten and 11 force peaks were identified in class 1 and class 2, respectively. Colored peaks and WLC curves show mean contour lengths (±SD) of the force peaks. Black lines indicate the total fit; dashed lines show baseline noise. Reproduced with permission from ref. Copyright 2015 Elsevier.

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  4. Effect of lipid composition on the folding of LacY. (a, b) Structural segments mapped to the secondary structure of WT LacY adopting the native (a) and the inverted (b) topology in POPE:POPG and POPG lipid membranes, respectively. Stable structural segments mapped in (a) and (b) were detected in the class 1 and class 2 force–distance curves, respectively. (a) Secondary structure model of LacY (PDB: 1PV7) mapped with 10 (class 1 force–distance curves) structural segments S1–S10 (native topology) and (b) with 11 (class 2 force–distance curves) structural segments S1–S4, S5*, S6*, S6**, S7*, S8, S9, S10 (inverted topology). Each mean contour length of a force peak class assigns the beginning of a structural segment (arrows pointing to aa). The numbers at arrows show the mean contour lengths of a force peak class (in aa), numbers in square brackets indicate the aa position counted from the C-terminal end, and numbers in parentheses give the aa position from the N-terminal end. Each of these numbers distinguishes the end of the previous and the beginning of a stable structural segment. Reproduced with permission from ref. Copyright 2015 Elsevier.

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  5. Single-molecule insertion and refolding of partially unfolded LacY into the phospholipid membrane. (a) Schematic of the refolding experiment of a single LacY using AFM-based SMFS. The tip of the AFM cantilever is attached to the C-terminus of LacY, and the cantilever is retracted to sequentially unfold the protein, leaving the last stable structural segment (S10, blue) embedded in the membrane. The resulting force–distance curve reflects the unfolding peaks of native LacY (see Figure ). (b) The AFM tip is then brought close to the membrane (∼10 nm) to relax the unfolded polypeptide. After 2 s of refolding time, the AFM tip is fully retracted to assess whether LacY remained unfolded, misfolded, or refolded structural segments. (b–d) Representative force–distance curves for LacY that remained unfolded (b), misfolded (c), or refolded partially (d). All experiments were performed in 50 mM KPi buffer, pH 7.2, at 25 °C. Reproduced with permission from ref. Copyright 2016 Springer Nature.

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  6. YidC suppresses misfolding and promotes proper insertion and folding of LacY into the phospholipid membrane. (a, b) Probability of unfolded LacY remaining unfolded (U), having misfolded (M), or having correctly refolded (F) in the absence (a) or presence (b) of YidC. (c) Probability of structural segments S1–S9 to misfold in the presence and absence of YidC. (d) Probability of proper insertion and folding of the structural segments under the same conditions. Differences of refolding experiments conducted in the absence and presence of YidC are considered significant. Significance tests with respect to WT LacY data are indicated: NS, P ≥ 0.01, * P < 0.01 and ** P < 0.001 as determined by two-tailed Z-test. Adapted with permission from ref. Copyright 2016 Springer Nature.

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  7. Model of the folding free-energy landscape of partially unfolded LacY in the presence of YidC. (a) In the absence of YidC, partially unfolded LacY misfolds with high probability. YidC prevents this misfolding and directs the partially unfolded LacY substrate through multiple folding intermediates toward folding its native state. (b) In the absence of YidC, the partially unfolded LacY inserts individual structural segments (S1–S10) into the membrane (red profile). However, folding is not completed and the majority of LacY substrates misfold after 5 s. YidC reduces misfolding and allows the unfolded LacY substrate to stepwise insert and fold its structural segments (S1–S10) into the membrane (green profile). Thereby, the structural segments insert and fold in random order until the folding of LacY has been completed. Adapted with permissions from refs. , Copyright 2016 Springer Nature and copyright 2017 American Chemical Society.

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  8. Schematic of the pull-and-paste assay to study the insertion and folding of single transmembrane proteins into a target membrane. The C-terminal end of a LacY (red) from a donor membrane is attached unspecifically to the tip of an AFM cantilever. Then, the LacY polypeptide is mechanically extracted and unfolded from the phospholipid membrane (1). The unfolded polypeptide is transferred to a target membrane containing the chaperone/insertase YidC (green) (2). At the YidC, the unfolded polypeptide is allowed to insert and fold into the membrane for a given time (3). With time, the unfolded LacY polypeptide progressively inserts and folds into the target membrane, ultimately adopting its native structure. Due to the temporary attachment of the C-terminal end to the tip, the end eventually slips off, allowing the tip to pick up another LacY for mechanical reconstitution (4). Reproduced with permission from ref. Copyright 2017 American Chemical Society.

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  9. YidC facilitates the integration and folding of extracted, unfolded LacY into the membrane. (a) AFM height image (topography) showing membranes containing LacY and YidC on mica in buffer solution. Scale bar: 1 μm. (b) Schematic of LacY (red) unfolding and extraction from a membrane by AFM-based SMFS. The AFM tip is first attached to the C-terminus, then the tip is retracted to mechanically extract and stepwise unfold the protein from the membrane. The resulting force–distance curve (red) shows characteristic unfolding peaks (segments S1–S10), matching the unfolding peaks recorded of native LacY (see Figure ). Gray WLC fits highlight each unfolding peak and step of LacY. (c) After extraction, the unfolded LacY polypeptide is laterally shifted 20–100 nm and held near (≈10 nm) a control membrane for 5 s. No insertion and refolding into the membrane is observed upon retraction. (d) In contrast, when LacY is moved to a YidC-containing membrane and held near the surface for 5 s, the force–distance curve (blue) shows native-like unfolding peaks, indicating the successful reinsertion and folding of the polypeptide into the native LacY structure. Reproduced with permission from ref.…

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  10. YidC- and SecYEG-mediated insertion differ kinetically and mechanistically. (a) Schematic of the insertion and folding experiment of a completely unfolded LacY polypeptide into a target membrane containing SecYEG. An AFM stylus unspecifically attaches the C terminus, mechanically unfolds and extracts LacY from the membrane, and then holds the unfolded polypeptide ∼5–10 nm above a SecYEG-containing phospholipid membrane for defined times. (b) Force–distance curve recorded in the presence of SecYEG after 10 s, showing the complete insertion and refolding of LacY. (c) Folding kinetics of LacY mechanically unfolded in the presence of YidC, SecYEG, or a SecYEG–YidC fusion complex. Colored linear fits represent insertion and folding rates of structural segments. (d) Force–distance curves showing single LacY polypeptides inserting and folding 2, 3, or 4 adjacent structural segments in the presence of SecYEG. WLC fits matching individual force peaks are shown in black, while the other WLC fits taken from the native LacY unfolding fingerprint are shown in gray. (e) Frequencies of detecting the insertion and folding of 2, 3, or 4 structural segments in the presence of of YidC, SecYEG, or a…

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  11. SecYEG and YidC assist the insertion and folding of LacY into a phospholipid membrane via distinct pathways. (a) YidC initiates LacY insertion and folding from any structural segment, after which the remaining segments (S1–S10) are then randomly inserted until folding is completed. (b) SecYEG, alone or together with YidC, also starts insertion and folding from any segment but proceeds sequentially. Dashed red arrows mark possible starting points of insertion, red double arrows indicate insertion and folding steps, and dashed black arrows show completion. Reproduced with permission from ref. Copyright 2019 The American Association for the Advancement of Science.

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  12. Unfolding and refolding of β-barrel membrane proteins. (a–d) Density plots of multiple superimposed force–distance curves recorded during mechanical unfolding of single (a) OmpA, (b) OmpG, (c) LamB, and (d) FhuA proteins from lipid membranes using AFM-based SMFS. WLC model curves of the contour lengths of the force peaks (numbers of amino acids) indicate the length of the structural segments being unfolded. (e) The insertion and folding pathway of β-barrel membrane proteins observed by single-molecule force spectroscopy. A β-barrel membrane protein is partially unfolded by applying a mechanical force to one terminus (1). The unfolded polypeptide is then relaxed (2). From the relaxed state, the unfolded polypeptide can refold in the lipid bilayer in a series of steps, which are shaped by the sequential insertion of individual β-hairpins (3). At any point along the folding pathway, complex β-barrel proteins can diverge from the main folding pathway and adopt a misfolded conformation. The final unfolding (5) determines whether the β-barrel membrane protein had attained a folded state or was trapped in a misfolded conformation (4). (f–h) Folding pathways and free-energy landscape of…