Pull-and-Paste of Single Transmembrane Proteins

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Pull-and-Paste of Single Transmembrane Proteins Tetiana Serdiuk, Stefania Anna Mari, and Daniel J. Müller Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b01844 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 23, 2017

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Pull-and-Paste of Single Transmembrane Proteins Tetiana Serdiuk,‡ Stefania A. Mari,‡ Daniel J. Müller,‡,* ‡

Department of Biosystems Science and Engineering, Eidgenössische Technische Hochschule (ETH) Zurich, 4058 Basel, Switzerland.

* [email protected] Fax ++41 61 387 3994

ACS Paragon Plus Environment

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ABSTRACT How complex cytoplasmic membrane proteins insert and fold into cellular membranes is not fully understood. One problem is the lack of suitable approaches that allow investigating the process by which polypeptides insert and fold into membranes. Here, we introduce a method to mechanically unfold and extract a single polytopic α-helical membrane protein, the lactose permease (LacY), from a phospholipid membrane, transport the fully unfolded polypeptide to another membrane and insert and refold the polypeptide into the native structure. Insertion and refolding of LacY is facilitated by the transmembrane chaperone/insertase YidC in the absence of the SecYEG translocon. Insertion into the membrane occurs in a stepwise, stochastic manner employing multiple co-existing pathways to complete the folding process. We anticipate that our approach will provide new means of studying the insertion and folding of membrane proteins and to mechanically reconstitute membrane proteins at high spatial precision and stoichiometric control thus allowing the functional programming of synthetic and biological membranes.

Keywords Membrane protein insertion and folding, folding pathway, mechanical reconstitution, single– molecule force spectroscopy, YidC insertase, atomic force microscopy


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In vivo the folding of cytoplasmic membrane proteins occurs in the context of vectorial translation of the freshly synthesized polypeptide, which exits the ribosome with its N-terminal end.4-6 However, translation is slow compared to folding and it takes ribosomes between ~ 5 seconds (bacteria) and ~ 25 seconds (eukaryotes) to synthesize a 100 amino acid (aa) long protein.7,8 Accordingly, the biogenesis of most polytopic membrane proteins depends critically on co-translational insertion and folding in the cytoplasmic membrane, which is facilitated by ribosome-translocon complexes.9 Basic elements of cytoplasmic membrane protein insertion and folding are conserved in pro- and eukaryotes.10-12 Bacteria employ SecYEG and YidC pathways to insert and fold α–helical membrane proteins.5,6,13 The translocon SecYEG and its homologs are critical for the insertion of proteins into the plasma membrane in bacteria and archaea, the endoplasmic reticulum in eukaryotes, and the chloroplast thylakoid membrane in plants.14 The membrane protein insertase YidC can either couple with the SecYEG pathway to facilitate folding and assembly of membrane proteins inserted by the Sec translocon or function via an independent pathway.15 In the YidC pathway, insertase activity catalyzes insertion, folding and assembly of proteins into the bacterial cytoplasmic membrane.13 Thus, YidC can function both as chaperone and insertase. This YidC pathway and those of its family members-Oxa1 in mitochondria and Alb3 in chloroplasts--is highly conserved.16,17 While chaperones suppress misfolding and aggregation of soluble proteins to assist folding,18,19 it is poorly understood how the insertion and folding of integral membrane proteins is assisted by chaperones, translocases and insertases.2 To characterize such assistance would need a method that allows observing the various folding intermediates a single polypeptide takes along its folding pathway towards the native membrane protein. For more than a decade atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to mechanically unfold single proteins from native or synthetic membranes.20,21 SMFS records a detailed fingerprint spectrum during protein unfolding that is sensitive to the fold, functional state, assembly, and environment of the membrane protein.22-26 The fingerprint spectrum measured under a given condition can be used to assign the fold and state of a single membrane protein. AFM-based SMFS has also been applied to partially unfold single membrane proteins and to directly observe their refolding at the level of secondary structural elements.27-30 It has been reported that after partial extraction from the membrane smaller proteins can self-insert and -fold in the membrane,27-30 whereas large and


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complex polytopic transmembrane proteins face difficulties to complete this task and misfold.31,32 In a cell, molecular chaperones are crucial for preventing unfolded polypeptides from misfolding and aggregating.2 A few SMFS studies describe the effect of chaperones on the folding trajectories of soluble proteins33-35 and of partially unfolded β-barrel outer membrane proteins36. Recently we observed that after partial extraction from the membrane, the lactose permease from Escherichia coli (LacY) can, with the assistance of YidC, insert and fold into the phospholipid membrane.32 As a model member of the major facilitator superfamily (MFS),37,38 LacY catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H+ (sugar/H+ symport) across the cellular membrane.39-41 LacY is a large polytopic membrane protein, which spans 12 mostly irregular transmembrane α-helices into pseudo symmetrical six helix bundles, which are connected by a relatively long cytoplasmic loop.42,43 In our previous experiments the partially unfolded LacY polypeptide started insertion and folding from Nterminal α-helices, which had been left in the membrane to initiate folding.32 Although this approach revealed insight into basic folding mechanisms, it could not access how a completely unfolded polypeptide from the outside of a membrane inserts and folds towards the native membrane protein. Here we introduce a nanomechanical assay to extract a single transmembrane protein from a membrane and to transport, insert and fold the protein into another membrane (Figure 1). Using the stylus of an AFM cantilever we attach the C-terminal end of the transmembrane transporter LacY from a donor membrane and apply a pulling force to unfold and extract the membrane protein. The completely unfolded LacY polypeptide is then moved with nanometer precision to another target membrane where it is held in close proximity to the surface (Figure 1). We observed that the polypeptide alone cannot insert and fold into the phospholipid membrane. However, in the presence of YidC we observe the stepwise insertion and folding of the polypeptide and, after a few seconds, the complete folding into the native LacY structure. Kinetic snapshots recorded along these folding pathways unravel the insertion and folding intermediates of the membrane protein. As the attachment of the C-terminal end to the AFM stylus is transient, LacY detaches quickly and the stylus is ready to pick up and extract another LacY from a donor membrane for insertion into a target membrane.

Preparing LacY and YidC for mechanical reconstitution To set up an extraction, transportation


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and insertion assay of LacY (Figure 1), LacY or YidC were reconstituted into phospholipid membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (PE)/1palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (PG) at a ratio of 3:1 (PE:PG)44 (Supporting Figure S1a). At this phospholipid composition, which is close to the composition of the E. coli membrane (70–80% zwitterionic PE and 20–25% anionic PG plus cardiolipin), LacY is fully functional and adopts its native topology exposing the N- and C-termini on the cytoplasmic surface.42,45 Co-translationally, the N-terminal end of a cytoplasmic membrane protein is targeted to translocons and insertases.4-6 To mimic this in vivo process we wanted to approach LacY to a membrane via its free N-terminal end while holding its C-terminal end with the AFM stylus. To enhance the probability of attaching the C-terminus of LacY by the AFM stylus, it was extended with a 36 aa long unstructured ‘polyGly’ polypeptide followed by an 8aa-long His-tag (His8-tag), which did not influence the structural and functional integrity of the transporter.26 To characterize the reconstituted membrane proteins morphologically, we adsorbed LacY or YidC proteoliposomes to mica in buffer solution and imaged them by AFM (Supporting Figure S1b,c). We also adsorbed the proteoliposomes to carbon grids and imaged them by TEM (Supporting Figure S1d,e). AFM topographs and TEM images showed singlelayered membrane patches. The AFM topographs revealed evenly distributed LacY or YidC particles within phospholipid membranes (Supporting Figure S1b,c). Next, we wanted to prepare donor membranes to mechanically extract and unfold single LacY polypeptides, which were then transported to target membranes embedding either LacY or YidC to characterize the insertion and folding of the polypeptide (Figure 1). We thus first adsorbed YidC proteoliposomes to mica in buffer solution where they appeared as single-layered membrane patches (Supporting Figure S2a). After having exchanged the buffer solution to halt the adsorption of YidC proteoliposomes, we identified the single layered YidC membranes by AFM imaging (Supporting Figure S2). Then, we adsorbed LacY proteoliposomes, which newly appeared as single-layered membranes.

Unfolding and extracting single LacY from membranes To extract a single LacY from the membrane by mechanically pulling the C-terminal end, we pushed the AFM stylus gently (700 pN for 500 ms) onto a LacY containing membrane to nonspecifically attach the artificially elongated C-terminal end (Figure 2a).26,46 Then the stylus was


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retracted from the membrane while recording the deflection of the AFM cantilever as a forcedistance curve (Figure 2b). These force-distance curves typically exhibited a saw-tooth-like pattern with 10 force peaks each indicating an unfolding step of the transporter.26 Repeating the experiment multiple times revealed a highly reproducible saw-tooth-like pattern (Figure 2). By fitting each of the 10 force peaks with the worm-like-chain (WLC) model46 we determined the contour lengths of the polypeptide segments unfolded in each step (Figure 2c,d). Controls showed that the polyGly extension of the C-terminus of LacY did not change the unfolding pattern compared to WT LacY (Supporting Figure S3).26 Importantly, the polyGly extension increased the probability of attaching the AFM stylus to the C-terminal end by a factor of ~ 10 (n = 2,974) and improved the detection of the very first unfolding force peaks detected in the force-distance curve.26 When applied mechanical pulling force to the elongated C-terminus of LacY, it was stretched until the first C-terminal segment unfolded (Figure 2e). As previously shown,32 this initial unfolding step elongated the polypeptide tethering the AFM stylus while the partially unfolded LacY molecule remained stably embedded in the membrane. Subsequent retraction of the stylus unfolded the transmembrane bundles stepwise in nine distinct structural segments formed by single or grouped α−helices and their connecting polypeptide loops.26 Thus, the force-distance curves recorded a characteristic ‘fingerprint’ spectrum for native LacY,32 with the 10 force peaks corresponding to the stepwise unfolding of 10 structural segments of LacY (Figure 2). The summed length of the force-distance curves corresponds to the contour length of the fully unfolded and stretched LacY polypeptide (Materials and Methods).26 It has been shown for LacY and for other membrane transporters and receptors that the force peaks of the native fingerprint spectrum are sensitive to the fold,25,36 functional state,47 ligand-binding,22,26,48 lipid composition,24,25 mutations and to their supramolecular assembly.49,50 As LacY inverts topology upon depletion of PE and becomes inactive,51 it was recently reported that the force peaks detected for the structural segments of native LacY change positions if LacY misfolds and can be used to assign structural segments changing their fold.25 Thus, the comparison of the native fingerprint spectrum of LacY with that recorded of a single LacY can distinguish correctly folded from unfolded and misfolded transporters.

LacY alone cannot insert and fold into phospholipid membranes


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Next, we wanted to test whether the fully unfolded and extracted LacY polypeptide can insert and fold into a phospholipid membrane. We thus unfolded and extracted a single LacY from the membrane and laterally moved the polypeptide with the AFM stylus by 20–100 nm where it was approached towards the membrane and held in close proximity (~ 10 nm) to the surface (Figure 3a-c). After 5 seconds, the stylus was retracted to detect whether the polypeptide interacted with the phospholipid bilayer. The force-distance curve recorded during retraction showed no interactions, indicating that the polypeptide did not insert into the membrane (Figure 3c). We repeated the single-molecule experiment 139,393 times. In none of these cases did we observe the extracted and unfolded polypeptide inserting and folding back into the native fingerprint spectrum of LacY (Supporting Figure S4). In rare cases (~ 0.005%) we detected that the polypeptide interacted with the membrane. However, the force-distance spectra showed force peaks occurring in a non-reproducible manner and which did not correlate with those observed in the native fingerprint spectrum (Supporting Figures S4 and S5a,b). We thus concluded that in these cases the unfolded polypeptide interacted with the membrane and misfolded, which is in agreement with previous reports that unfolded membrane proteins outside the membrane and in the absence of chaperones or chaotropic agents misfold and aggregate.2,52 This finding differed considerably from previous SMFS experiments showing that LacY after partial extraction from the membrane could reinsert and refold some but not all structural segments into the lipid membrane.32 Obviously, this difference highlights that it is much easier for a polypeptide, which has been already partially inserted into the membrane to conduct insertion and folding attempts compared to a completely unfolded and extracted polypeptide. In summary, our experiments showed that the extracted and unfolded LacY polypeptide cannot self-insert into the phospholipid membrane and fold its native structure.

YidC supports insertion and folding of LacY into membranes It has been previously suggested that YidC requires the SecYEG complex for the insertion of the LacY polypeptide.53-55 We wondered whether YidC alone could assist the insertion and folding of LacY into the membrane. As a control, we first pushed the AFM stylus onto a YidC proteoliposomes, to attach the terminal end of a single YidC and upon withdrawal of the stylus to mechanically unfold an insertase (Supporting Figure S6). The force-distance spectra recorded from 107 of such attempts showed a longer and markedly different force peak pattern compared


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to the fingerprint spectrum of native LacY. Thus, force-distance patterns recorded for YidC and LacY are clearly distinguishable. Next, we extracted and unfolded a single LacY from the Cterminal end but now approached the unfolded polypeptide to a membrane embedding the chaperone/insertase YidC (Figures 1 and 3a,b,d). As described above, the unfolded LacY polypeptide was kept in close proximity (~ 10 nm) to the membrane surface where it was allowed to insert and fold for 5 seconds. Finally, retraction of the AFM stylus showed a distinct force-distance pattern (Figure 3d). To interpret the pattern recorded, the native fingerprint spectrum of LacY was used as template. It has been previously shown that the force peaks of the force-distance pattern considerably change position if the LacY polypeptide misfolds.25,32 However, because the unfolding force peaks of the refolded LacY polypeptide occurred at positions detected in the native fingerprint spectrum of LacY (Figure 2c and Supporting Figure S5c,d), the polypeptide was interpreted as having correctly inserted and folded structural segments into the phospholipid membrane. Moreover, the refolded polypeptide established all characteristic force peaks detected for native LacY and because this native fingerprint spectrum is specific for the fold and functional state of LacY,25,26 we concluded that the polypeptide completed insertion and folding into the native LacY structure. Intrigued by the possibility that YidC could insert the completely unfolded and extracted LacY polypeptide into the phospholipid membrane we repeated the insertion and folding experiments over the time course of several weeks (Supporting Figure S7). For each experimental day we prepared fresh samples and AFM cantilevers. Taken together we performed 136,741 single-molecule experiments at 5 second refolding time. The majority of force-distance curves, which detected refolding events (89%, n = 37), showed only force peaks of the native fingerprint spectrum of LacY and thus detected the insertion and folding of native structural segments (Supporting Figure S7). The average force required to unfold a stable structural segment folded by the LacY polypeptide was 88.2 ± 33.1 pN (mean ± SD; n = 198), indistinguishable to that required to unfold a structural segment from native LacY (88.3 ± 32.1 pN; n = 298). Thus, LacY inserted and folded with the help of YidC exhibited the same mechanical stability as native LacY. To further investigate whether non-specific interactions could play a role in LacY insertion and folding, we performed control experiments in the presence of BSA or lysozyme in which the completely unfolded and extracted LacY polypeptide was allowed to fold into a phospholipid membrane in the absence of YidC. In more


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than 200,000 single-molecule experiments we could not observe a single correctly inserted and folded LacY polypeptide (Supporting Figure S8). Similarly, we observed that the presence of other LacY molecules in the membrane did not support insertion and folding of the LacY polypeptide (Figure 3). We thus concluded that YidC guides the insertion and folding of a completely unfolded and extracted LacY substrate in a specific manner towards its native structure. Our finding was surprising, since in vivo the insertion of LacY is thought to be facilitated by the SecYEG translocon while the folding is assisted by YidC.53,55 We thus questioned how YidC alone could assist the insertion and folding of the LacY polypeptide.

YidC supports the stepwise insertion and folding of LacY to completion To investigate how YidC assists the insertion and folding process of a fully extracted polypeptide, we allowed single unfolded and extracted LacY polypeptides to fold into the YidC membrane for times ranging from 1–5 seconds. After the folding time elapsed the AFM stylus was retracted to detect the structural segments the LacY polypeptide inserted and folded (Figure 4a and Supporting Figure S9). Comparison of the unfolding spectrum of the folded polypeptide with the fingerprint spectrum of native LacY assigned the correctly folded structural segments (Supporting Figure S5). We observed that with increasing folding time the LacY polypeptide folded an increasing number of structural segments (Figure 4b) until completing the folding of the native LacY structure (Figure 4a and Supporting Figure S9). This result highlighted that the polypeptide could not complete insertion and folding in short folding times whereas after 5 seconds it could. However, after 5 seconds only ~ 12% of the LacY molecules completed insertion and folding (n = 33). Thus, it requires an even longer folding time to guide every unfolded LacY polypeptide towards the native LacY structure. Furthermore, comparison with the fingerprint spectrum of native LacY showed that the LacY polypeptide in each experiment inserted structural segments in a random order (Figure 4a and Supporting Figure S9). The probability distribution of structural segments inserting at long refolding times was uniform (at 3 seconds refolding time, p = 0.1699, Chi-square test), underlining this stochastic effect (Figure 4c).

LacY inserted by YidC remains stably folded in the membrane We wondered how we could use our assay to deliver the unfolded LacY polypeptide to a target


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membrane and whether the folded LacY remains stably embedded in the membrane. When mechanically pushing the AFM stylus onto a polypeptide it attaches non-specifically but sufficiently strong to allow the mechanical unfolding of water-soluble or membrane proteins.56,57 It has been reported that this non-specific attachment is transient and after a certain time (in the range of seconds) the polypeptide slips from the tip.46,57 Indeed, in ~ 94% of all cases (n = 1,091), the C-terminal end of LacY detached from the AFM stylus within 5 seconds refolding time.32 We thus decided to mechanically extract one LacY after another from the phospholipid membrane, to bring them into close proximity to the target membrane embedding YidC and to wait for a longer time period (Figure 5a,b). After a waiting time of 10 seconds we retracted the AFM stylus and detected no interactions (Figure 5c,d). Thus, the unfolded LacY polypeptide may have inserted and folded into the target membrane and slipped from the AFM stylus or did not interact with the membrane. We repeated the single-molecule experiments by operating the AFM automatically over the time course of two days at controlled room temperature and buffer solution. All together we unfolded and extracted 43 LacY polypeptides from donor membranes, all of which were brought into close proximity (~ 10 nm) to the YidC target membrane. Finally, a clean AFM stylus was brought into contact with the same target membrane in which we had attempted to insert LacY molecules. The majority of force-distance spectra recorded for proteins unfolded from this membrane matched the unfolding spectrum of YidC (Supporting Figure S6). However, in some attempts we detected force-distance spectra that showed the specific force peak pattern of native LacY (Figure 5e,f). We can thus report the successful mechanical reconstitution of LacY to the YidC target membrane at which LacY remained stably embedded.

Here we have introduced a SMFS-based pull-and-paste assay to mechanically extract, transport and reconstitute transmembrane transporters into target membranes. In principle, using this assay to reconstitute membrane proteins one could equip synthetic or biological membranes with novel functions. On another hand, our assay allows studying the insertion and folding processes of completely unfolded and extracted polypeptides at near physiological conditions into their native structure within a membrane. Thereby, the AFM stylus roughly mimics the role of a ribosome holding the N-terminal end of the LacY polypeptide in close proximity to the membrane, while constraining the C-terminus. In the absence of YidC, the unfolded LacY


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polypeptide was unable to self-insert and -fold into the phospholipid membrane. In the presence of YidC, however, we observed the successful insertion and folding of completely extracted and unfolded LacY polypeptide. This result indicates that initialization of LacY insertion into the phospholipid membrane is prevented by an energy barrier that is too high to be overcome by the unfolded LacY polypeptide alone (Figure 6a). This finding also reflects the fact, that the insertion of membrane proteins into cellular membranes does not occur spontaneously in live cells, and requires the help specific insertases, translocases, or/and chaperones. However, YidC considerably lowers this free energy barrier such that LacY can start to insert and fold structural segments into the membrane (Figure 6a). Once this insertion and folding has been initiated the stepwise insertion and folding proceeds until the polypeptide has completed the folding of native LacY (Figure 6a,b). In the presence of YidC it appears as if all structural segments show quite similar probability to insert and fold. This equality leads to structural segments inserting and folding in a stochastic order, without any obvious preference (Figure 6b). Thus, one may assume that collectively these interactions find many pathways to fold the polypeptide towards the native LacY structure. In light of our observations, one could propose a model where the key function of YidC is to initialize the insertion and folding of a first structural segment of the unfolded polypeptide into a membrane. During the stepwise insertion and folding of the following structural segments the chaperoning function of YidC prevents the unfolded and not yet inserted polypeptide stretches from misfolding.32 With proceeding number of structural segments inserted the interactions between these segments drive their folding into the tertiary structure (Figure 6). Our finding of the stepwise and stochastic insertion and folding towards the native structure is supported by previous single-molecule experiments, which observed the same effect for partially extracted and unfolded LacY polypeptides.32 Both the SecYEG translocon and YidC chaperone/insertase were found to be important for LacY insertion and folding,53,55 however, our assay shows that solely YidC can facilitate the insertion and folding of the LacY polypeptide into its native structure from completely unfolded and extracted state. This finding is in agreement with previous reports showing that YidC can insert short transmembrane stretches into lipid membranes.59 Exhibiting very different structural features SecYEG and YidC are thought to support the insertion of membrane proteins by lowering the thermodynamic barrier for an unfolded protein chain to insert into the amphiphilic


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lipid membrane and to achieve its native fold.4,13 Recent experimental insights suggest that the signal recognition particle (SRP)-pathway delivers membrane proteins co-translationally to two distinct integration sites of the E. coli membrane which are either SecYEG or YidC translocases.54 However, what directs an unfolded polypeptide to take the SecYEG, YidC or SecYEG/YidC pathway is not yet clear. Our single molecule assay might help to answer this question as it can be readily applied to study membrane protein insertion and folding by SecYEG, YidC, eukaryotic Sec61, TRAP, TRAM or any other reconstituted insertase alone or in combination with soluble chaperones. In this regard, our experiments may be seen as a first milestone towards studying the mechanisms by which YidC, SecYEG and the SecYEG/YidC complexes insert and fold membrane proteins and thus could shed light on the general mechanisms of membrane protein folding, which are conserved from E. coli to humans.


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LacY and YidC engineering, expression and purification. The C-terminal end of (wild-type) WT LacY was elongated with a 36 amino acids (aa) long ‘polyGly’ extension followed by a His8-tag [GSM(G11)EAVEEAVEEA(Gly11)S(His8)] using QuikChange II PCR and plasmid pT7-5/LacY as template. PolyGly LacY and WT LacY were purified from E. coli XL1-Blue (Stratagene) transformed with pT7-5 plasmids harboring given mutant genes by using Co(II) affinity chromatography as described.60 After elution from the Co(II)-Talon column LacY was concentrated and washed with 50 mM sodium phosphate (NaPi), pH 7.5 and 0.01% (w/w) dodecyl-β-D-maltopyranoside (DDM, Maumee) on an Amicon Ultra-15 concentrator with a 30 kDa cut-off (Millipore). WT YidC carrying a His10-tag on the C-terminus was cloned in pT7-7 plasmid and expressed and purified similar to LacY except that the expression strain used was E. coli BL21 (DE3) and the DDM concentration used was 0.03%. After elution from the Co(II)-Talon column YidC was concentrated and washed with 150 mM NaCl, 50 mM sodium phosphate (NaPi), pH 7.5 and 0.03% (w/w) DDM on an Amicon Ultra-15 concentrator with a 30 kDa cut-off. Prepared proteins were at least 95% pure as shown by staining after sodium dodecyl sulfate polyacrylamide (SDS) gel electrophoresis (Supporting Figure S1). LacY or YidC were reconstituted into proteoliposomes with 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine (PE, Avanti Polar Lipids) and 1-palmitoyl-2-oleoyl-sn-glycero3-phospho-rac-(1-glycerol) (PG, Avanti Polar Lipids) (ratio 3:1) using the dilution method.44 Briefly, protein concentrations of LacY or YidC were dissolved in 0.01% DDM solution and mixed with PE:PG phospholipids dissolved in 1.2% octyl glucoside (OG, Maumee) to gain a lipid-to-protein ratio of 5 (w/w). The mixture was stored for 20 minutes on ice and then quickly diluted 50-fold in 50 mM NaPi, pH 7.5. Proteoliposomes were collected by centrifugation (100,000g) for 1 h, suspended in the same buffer and flash frozen in liquid nitrogen. AFM-based single-molecule force spectroscopy (SMFS). The same AFM (Nanowizard II Ultra, JPK Instruments AG, Berlin, Germany) equipped with an 850 nm laser detection system was used for all SMFS experiments. To reduce thermal drift, the AFM was placed in a temperature stabilized room (25 ± 0.1°C). To reduce acoustic noise and drift caused by airflow the AFM was placed into a homebuilt acoustic box. To compensate the evaporation of buffer,


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the volume of buffer and electrolyte concentration were controlled and kept constant. All AFM experiments were done in SMFS buffer (50 mM potassium phosphate (KPi), pH 7.2) and performed with Si3N4 cantilevers (OMCL RC800PSA, Olympus) having a nominal spring constant of 0.05 N·m–1. Before and after of each experiment cantilevers were calibrated in SMFS buffer using the equipartition theorem.61 Single-molecule unfolding experiments and data analysis. SMFS unfolding experiments and data analysis were performed as described.26 Briefly, PE:PG (ratio 3:1) proteoliposomes containing reconstituted LacY or YidC were adsorbed to freshly cleaved mica in SMFS buffer for 20 min. To remove weakly attached proteoliposomes the samples were then rinsed with SMFS buffer several times. At the beginning of each experiment the reconstituted membrane proteins were imaged by AFM (Supporting Figure S2a,b).46 On average, AFM imaging of the reconstituted LacY or YidC revealed approximately the same density of reconstituted membrane proteins (Supporting Figure S1b,c). To perform SMFS, the AFM stylus was pushed to the membrane with the contact force ~ 700 pN for 500 ms. The probability of the AFM tip to nonspecifically attach the ‘polyGly’ elongated C-terminal end of LacY (~ 0.1%, n = 2,974) was ~ 10-times higher than the probability to attach the non-elongated C-terminal end of WT LacY.26 A completely unfolded and stretched LacY [417 aa extended by a 36 aa long polyGly tail and a His8-tag] exhibits a contour length 461 aa and shows the force peak patterns extending to a distance of ~ 120 nm (Figure 2). Thus, to fully unfold and extract the LacY from the membrane the cantilever was retracted by ≥ 190 nm at constant speed (0.7 µm·s–1). A completely unfolded and stretched YidC polypeptide [551 aa extended by an His10-tag] shows a contour length 561 aa and extends to ~ 150 nm. To characterize the unfolding of YidC, we thus selected force-distance curves showing force peak patterns extending to >> 130 nm to ensure that only curves recording the full unfolding of YidC from pulling one terminal end were analyzed (Supporting Figure S6). The deflection sensitivity and force offset was corrected for every force-distance curve using the standard AFM data processing software (JPK Instruments, software version 5.0.12). To fit the individual force peaks of the force-distance curves the worm-like-chain (WLC) model with a persistence length of 0.4 nm and contour length of 0.36 nm per aa was applied.46,62 For each unfolding force peak the WLC provided the contour length (number of aa) of the polypeptide unfolded and stretched. To analyze the contour length at which force peaks from all


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experiments were detected histograms of these contour lengths were fitted with the Gaussian mixture model (Figure 2d and Supporting Figure S6b).63 In this model, each i-th detected contour length li belongs to one force peak class s = 1, …. , M with probability πs or originates from background noise with probability π0. We detected 10 force peaks classes for the fingerprint spectrum of native LacY and 8 force peak classes for the fingerprint spectrum of YidC. The mean contour length µs for a given force peak class s was described by a Gaussian distribution with variance σs2. The probability density f of li can be shown as a mixture of Gaussians and background noise with weights πs and π0. f = ∑ , , +

(1)

ϕ(li, µs, σs2) is the probability density of the Gaussian distribution and g(li) the background noise. To find parameters of the Gaussian mixture model (π, µ, σ2) the expectation optimization algorithm was used. The most probable force peak class si was assigned to any given contour length li with the Bayes classifier by setting: = , , ,

(2)

Every detected contour length was assigned to a force peak class. For LacY, each of the force peak classes was mapped to the secondary structure (Figure 2e).26 The polyGly and His8-tag elongation of the C-terminal end was accounted. The thickness of the membrane was taken into account if the force peak class located the beginning or end of a stabilizing structural segment on the mica-facing side of the membrane or within the lipid membrane.46 Single molecule extraction, insertion and folding. First, proteoliposomes with YidC reconstituted in PE:PG (3:1) were adsorbed to freshly cleaved mica in SMFS buffer for 20 min. Several cycles of gentle rinsing of the sample with SMFS buffer were performed to remove the weakly attached proteoliposomes. Subsequent AFM imaging identified the membrane patches with reconstituted YidC (Supporting Figure S2a). Then, proteoliposomes containing LacY reconstituted in PE:PG (ratio 3:1) were applied to the same sample for 30 min. After given time passed for adsorption, the sample was rinsed with SMFS buffer to remove weakly attached proteoliposomes. AFM images identified the newly adsorbed LacY and the before ahead adsorbed YidC membrane patches. After having fully unfolded and extracted a single LacY


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from the LacY membrane patch, we brought the extracted polypeptide into close proximity (~ 10 nm) to the membrane surface of either the same LacY membrane or to a membrane patch containing YidC. At this close proximity, the LacY polypeptide was held in the relaxed state for a given time (1–10 seconds) to allow insertion and folding. Force curves indicating that within this time period the AFM stylus has touched the membrane (i.e., thermal drift) were discarded from further analysis. After the folding time passed, the AFM stylus was fully retracted to detect whether the LacY polypeptide inserted and folded into the membrane. The force-distance curve recorded during this retraction was inspected to reveal which structural segments of LacY inserted and folded. To exclude rare (~ 0.005%) non-specific interactions of the unfolded LacY with the membrane only force-distance curves showing a pattern > 10 nm were accounted. Correct insertion and folding was accounted if this second force curve exhibited only force peaks from the native fingerprint pattern of LacY (Figure 2 and Supporting Figure S5). In case of detecting no force peaks we assumed the LacY polypeptide to having not inserted and folded. In case of force peaks were detected at positions different than those detected for the native fingerprint spectrum, the LacY polypeptide was concluded to having misfolded (Supporting Figure S5). Control of LacY and YidC reconstitution. Successful reconstitution of either LacY or YidC in PE:PG membranes was demonstrated by AFM imaging (Supporting Figure S1) and SMFS unfolding experiments (Figure 2 and Supporting Figure S6). SMFS was performed on at least eight different membrane patches with reconstituted LacY or YidC. The superimposition of recorded force-distance curves revealed the unique force peak pattern of either LacY or YidC. The force peak pattern of this superimposition served as the native fingerprint spectrum of LacY or YidC. Insertion and folding experiments, data analysis and classification. Force-distance curves recorded in our experiments allowing the insertion and folding of the unfolded LacY polypeptide were analyzed as follows: The force peaks in force-distance curves detected of inserted and folded LacY were fitted using the WLC model to determine their contour lengths. The comparison of these force peaks with those detected in the fingerprint spectrum of native LacY allowed the insertion and folding experiment being classified as described in the manuscript and Supporting Figure S5. Briefly, a force-distance curve was classified to present a LacY substrate


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remaining unfolded if no interactions were detected after the folding time passed. A forcedistance curve was classified to present a LacY substrate having misfolded if it detected at least one force peak at a position not matching the mean ± s.d. of a force peak position detected in the native unfolding force peak pattern of LacY (Figure 2). A force-distance curve was classified to present a LacY having inserted and folded one or more structural segments if it detected at least one force peak at a position matching the mean ± s.d. of a force peak position detected in the native unfolding force peak pattern of LacY (Figure 2). Force-distance curves detecting insertion, folding and misfolding events were classified to present a misfolded LacY. To test whether unspecific interactions play a role in determining the outcome of the insertion and folding experiments, LacY has been allowed to insert and fold as described above but in the presence of either 1 µM BSA (Sigma-Aldrich) or 1 µM lysozyme (Fluka Analytical). All insertion and folding experiments were analyzed following the same procedure and classified applying the same criteria described above. Statistical data analysis. The set of 309 insertion and folding experiments in the presence of YidC was collected at different insertion times ranging from 1 to 5 seconds. At least 30 folding events were analyzed for each given insertion and folding time. No correct reinsertion was detected in each of ~ 100,000 attempts for each control condition (LacY, LacY & BSA, LacY & Lysozyme). The set of 69 single reinsertion events recorded at 3 seconds insertion time and representing 252 folded segments was analyzed for mapping a probability of structural segments to reinsert (Figure 4c). Chi-square test was used to check if the probability distribution of the inserted structural segments was uniform. Statistical analysis was performed using R. Data availability. The authors declare that the main data supporting the findings of this study are available within the article and its Supporting Information files.

ASSOCIATED CONTENT Supporting Information. SDS gel, AFM topographs and TEM images of LacY and YidC reconstituted in PE:PG (3:1) phospholipid membranes (Figure S1). AFM topographs showing co-adsorption of phospholipid membranes embedding either YidC or LacY (Figure S2). SDS gel and SMFS of polyGly elongated and LacY (Figure S3). Controls showing no unaided LacY


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reinsertion (Figure S4). Criteria of classifying misfolding and refolding events of LacY (Figure S5). SMFS of reconstituted YidC (Figure S6). Examples of LacY reinsertion in the presence of YidC at 5s refolding time (Figure S7). Non-specific controls of LacY insertion (Figure S8). Kinetic snapshots of LacY folding intermediates recorded at refolding times ranging from 1 to 5 seconds (Figure S9).

AUTHOR INFORMATION Corresponding Author. *Email: (D.J.M) [email protected]

Author Contributions. T.S. and D.J.M. designed the experiments. T.S. performed the AFMbased SMFS experiments and S.A.M. recorded AFM images. All authors analyzed experimental data and wrote the paper.

Competing financial interests. The authors declare no competing financial interests.

Acknowledgments We thank H.R. Kaback for providing reconstituted LacY and YidC samples and for constructive discussion and support of the project, R.E. Dalbey for providing plasmid pT7-7 encoding YidC with a His10-tag at the C-terminus, and S. Weiser, D. Balasubramaniam and J. Sugihara for reconstituting the LacY and YidC samples, BioEM Lab facility of the University of Basel for providing TEM images, A. Kuhn, N. Beerenwinkel, R. Newton, and J. Thoma for encouraging and constructive comments. This work was supported by the Eidgenössische Technische Hochschule Zürich (to D.J.M.), the Swiss National Science Foundation (Grant 205320_160199 to D.J.M.), the National Center of Competence in Research “NCCR Molecular Systems Engineering” (to D.J.M.) and the European Union Marie Curie Actions program through the ACRITAS Initial Training Network (FP7-PEOPLE-2012-ITN, Project 317348 to D.J.M.).


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Figure 1. Cartoon of the pull-and-paste process of single transmembrane proteins. From a donor membrane a single transmembrane protein LacY (red) is picked up from the elongated C-terminal end using the stylus of an AFM cantilever (1) and mechanically unfolded and extracted from the phospholipid membrane (2). Then, the unfolded LacY polypeptide is transported to a target membrane embedding the chaperone/insertase YidC (green) (3). After a certain folding time the polypeptide inserts and folds stepwise into the target membrane until folding into the native LacY structure has been completed. Because of the transient attachment to the stylus the C-terminal end of the polypeptide slips from the stylus and the stylus can be taken to mechanically pick up and extract the next LacY for reconstitution into a target membrane (4). Figure 2. Mechanical unfolding by SMFS reveals the fingerprint pattern and unfolding pathway of native LacY. (a) Schematics of the unfolding of a single LacY from the phospholipid membrane using the stylus of the AFM cantilever. First, the AFM stylus is pushed onto LacY (PDB 1PV7) to facilitate the nonspecific attachment of the elongated C-terminal end (polyGly LacY). Then, the cantilever is retracted to apply mechanical force to the protein. Exposed to mechanically pulling force LacY unfolds stepwise until completely extracted from the membrane.26 (b) Force-distance curve recorded upon mechanical unfolding of single LacY from the lipid membrane. (c) Superimposition of 705 force-distance curves each recorded upon mechanically unfolding a single LacY from PE:PG (ratio 3:1) membranes. Each force peak of the density plot was fitted with the worm-like-chain (WLC) model (grey curves)46,62 to obtain the contour length of the unfolded and stretched polypeptide. The mean contour length (in aa) is indicated for each WLC curve. (d) Contour length histogram of all force peaks detected in 705 force-distance curves (grey bars) fitted with a Gaussian mixture model (black line).63 Each individually colored Gaussian distribution provides the mean contour length of a force peak class (given in aa ± s.d. at the top of each Gaussian distribution). Ten force peak classes were identified for LacY. The black solid line shows the sum of weighted contour lengths for all force peak classes and the dashed line represents the uniform baseline noise. Each mean contour length of a force peak class allocates the ending of the previously unfolded structural segment and the beginning of the next structural segment to be unfolded. (e) Ten structural segments (S1-S10) were mapped to the secondary structure of LacY. Numbers give the mean contour length of a force peak class and stars indicate beginning and ends of structural segments. Figure 3. The chaperone/insertase YidC facilitates the insertion and folding of the fully unfolded and extracted LacY polypeptide into the membrane. (a) AFM image of LacY and YidC membranes adsorbed to mica in buffer solution (taken from Supporting Figure S2b). Scale bar, 1 µm. (b) Schematics of unfolding and extracting a single LacY (red) from the phospholipid membrane using the stylus of the AFM cantilever. First, the AFM stylus is pushed onto LacY to facilitate the non-specific attachment of the elongated C-terminal end. Then, the cantilever is retracted to apply mechanical force to the protein. Exposed to mechanically pulling force LacY unfolds stepwise until completely extracted from the membrane.26 The force-distance curve (red) recording this unfolding and extraction process shows force peaks of the fingerprint spectrum of native LacY (Figure 2). For comparison WLC curves (grey) fitting the unfolding force peaks of the native fingerprint spectrum are shown. Each force peak detects an unfolding step of LacY and after the last unfolding step the polypeptide has been extracted from the membrane (> 120 nm). The ten structural segments S1–S10 unfolded by each force peak are indicated at the top of each WLC curve. (c) After unfolding and extraction the LacY polypeptide is laterally shifted by ~ 20–100 nm and then approached close to the membrane surface (~ 10 nm) were it is kept for 5 seconds


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to allow the polypeptide to reinsert and fold. After this time passed the stylus is completely retracted to characterize whether the polypeptide remained unfolded, misfolded or folded structural segments. The force-distance curve (blue) shows no interactions of the unfolded polypeptide with the membrane. (d) After unfolding and extraction the LacY polypeptide is laterally shifted to the YidC membrane and then approached close to the membrane surface (~ 10 nm) were it is kept for 5 seconds to allow reinsertion and folding. After this time passed the stylus is completely retracted to characterize whether the LacY substrate remained unfolded, misfolded or folded structural segments. The force-distance curve (blue) recorded shows force peaks of the fingerprint spectrum of native LacY. For more examples see Supporting Figures S4 and S7. Figure 4. YidC assists the LacY polypeptide to insert and fold along multiple co-existing pathways. (a) Force-distance curves recorded after transporting fully unfolded and extracted LacY polypeptides to a YidC membrane and allowing the polypeptides to insert and fold for times varying from 1 to 5 seconds (experiments conducted as described in Figure 3). Each force-distance curve records the unfolding of a single folded LacY polypeptide. WLC curves (grey and black) taken from the fingerprint spectrum of native LacY (Supporting Figure S2) assign the unfolding force peaks to the structural segments S1–S10 and thus allow assigning the structural segments the polypeptide folded with assistance of YidC. WLC curves of the native fingerprint spectrum matching the force peaks in terms of mean ± s.d. are colored black, WLC fits not matching any force peak are colored grey. Examples are taken from a set of 309 folding experiments. For more examples see Supporting Figure S9a,b. (b) Average number of structural segments inserted per substrate in dependence on the folding time (number of analyzed folding events n ≥ 30 per time point). (c) Probability of structural segments S1–S10 folding per substrate at 3 seconds folding time (n = 69 folding events, n = 252 refolded structural segments detected) is undistinguishable from a uniform distribution (p = 0.1699, Chi-square test). Error bars indicate s.e.. Figure 5. Mechanically controlled extraction, transport, insertion, folding and deposition of single lactose permeases from a donor into a target membrane. (a) Schematic and (b) force-distance curve recorded of the mechanical unfolding and extraction of a single lactose permease from a donor membrane. The forcedistance curve (red) shows force peaks of the unique fingerprint spectrum of native LacY. The structural segment S1–S10 unfolded in each force peak is indicated at the top of each WLC curve taken from the native fingerprint spectrum (Figure 2). (c) After unfolding and extraction of the LacY polypeptide, the AFM stylus is moved to another membrane containing YidC and to which surface it is kept in close proximity (~ 10 nm) for 10 seconds. This time is sufficient to allow the insertion and folding of the polypeptide at the target membrane. Because the non-specific attachment of the elongated C-terminal end of the LacY polypeptide to the AFM stylus is transient46 it slips off the stylus after 5 seconds refolding time with 94% probability (n = 1,091). (d) Accordingly, the force-distance curve (blue) recorded after delivery of the LacY polypeptide measured no interaction forces. (e) After repeating above described single-molecule experiment for many hundred times over the time range of 45 hours, the AFM stylus is pushed onto the target membrane to facilitate the non-specific attachment of a delivered membrane protein. (f) Upon retraction of the cantilever the characteristic fingerprint spectrum of native LacY is recorded indicating the successful and kinetically stable long-term insertion of LacY. Figure 6. YidC lowers the free energy barrier to insert and fold the unfolded LacY into native structure. (a) Schematic model of a high free energy barrier preventing the self-insertion of LacY into the phospholipid membrane and model of the free energy landscape of YidC-assisted insertion and folding of the LacY polypeptide. YidC lowers the energy barrier to facilitate the stepwise insertion and folding of


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the LacY polypeptide and prevents the polypeptide from misfolding until folding has been completed. (b) YidC employs variable folding pathways to insert and fold LacY. Two examples of LacY folding pathways are shown in black and red. The structural segments S1–S10 establish 10 insertion and folding steps that are required to complete folding of LacY. Insertion and folding process proceeds through all 10 structural segments stepwise, in random order, until LacY native fold is completed.


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Figure 1 58x40mm (300 x 300 DPI)


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Figure 3 123x87mm (300 x 300 DPI)


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Figure 4 134x127mm (300 x 300 DPI)


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Figure 6 107x65mm (300 x 300 DPI)


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For TOC only 39x19mm (300 x 300 DPI)


Pull-and-Paste of Single Transmembrane Proteins

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