Single-molecule dynamics reveal ATP binding alone powers substrate translocation by an ABC transporter

Domain: biology
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
ATP-binding cassette (ABC) transporters are molecular machines involved in diverse physiological processes, including antigen processing by TAP, a key component of adaptive immunity. TAP and its bacterial homolog TmrAB use ATP to translocate peptides across membranes, yet the precise mechanism linking ATP binding to substrate movement remains unclear. Here, we employ a single-molecule FRET sensor to visualize single translocation events by individual ABC transporters and thereby overcome the limitations of ensemble averaging. This approach reveals that substrate transport is driven by a conformational switch from the inward- to the outward-facing state. Using a slow-turnover TmrAB variant, we demonstrate that ATP binding alone, even in the absence of Mg2+, is sufficient to drive a single round of peptide translocation. Cryo-EM structures of wild-type and slow-turnover TmrAB show that ATP binding induces the outward-facing conformation even without Mg2+. In wild-type TmrAB, this conformational transition supports a single translocation event, whereas Mg2+-dependent ATP hydrolysis is required to reset the transporter. These findings establish a direct mechanistic link between ATP binding and substrate translocation at single-molecule resolution and provide insight into the catalytic cycle of ABC transporters.

Introduction
ABC transporters represent the largest family of primary active transport proteins, conserved across all domains of life1–3. Using the energy from ATP binding and hydrolysis, they move a broad spectrum of substrates, e.g., ions, amino acids, lipids, vitamins, proteins, and peptides, against concentration gradients across biological membranes. Because of their central roles in human diseases, including multidrug resistance, bacterial pathogenicity, and genetic disorders, a detailed understanding of their molecular mechanisms is of significant clinical importance4–6.

ABC proteins are classified according to conserved sequence features within their nucleotide-binding domains (NBDs)7 and structural organization of their transmembrane domains (TMDs)8. Among type IV ABC transporters, P-glycoprotein (P-gp, MDR1) and heterodimeric transporters such as the transporter associated with antigen processing (TAP1/2) and its prokaryotic homolog, the Thermus thermophilus multidrug resistance protein A and B (TmrAB), are notable examples8–10. TAP plays a pivotal role in adaptive immunity by transporting antigenic peptides from the cytosol into the endoplasmic reticulum, where they are loaded onto major histocompatibility complex (MHC) class I molecules for immune surveillance11,12. TmrAB, a structural and functional homolog of TAP with overlapping substrate specificity, can rescue TAP deficiency in human cells13. Cryogenic electron microscopy (cryo-EM) analyses have captured multiple conformational states of TmrAB during turnover14. However, ensemble approaches cannot resolve the order or kinetics of individual substrate-translocation steps, leaving key mechanistic questions open.

A central challenge in understanding ABC transporter energetics is disentangling the contributions of ATP binding, ATP hydrolysis, and the involvement of Mg2+ as a cofactor. It remains unclear whether ATP binding alone is sufficient to initiate substrate translocation or whether hydrolysis is also required15,16. The role of Mg2+ remains controversial: while some studies suggest it is essential for ATP binding17, others indicate that NBD dimerization can occur in its absence18–20. We hypothesized that ATP binding alone drives single-substrate translocation, whereas Mg2+-dependent ATP hydrolysis is necessary to reset the transporter from the outward-facing (OF) to the inward-facing (IF) state.

Heterodimeric type IV ABC transporters, including TAP1/2 and TmrAB, add another level of complexity due to their intrinsic functional asymmetry9,21–23. This asymmetry complicates mechanistic interpretation, particularly with respect to the efficiency and modulation of the transport cycle—areas that are not yet fully understood. Resolving these issues requires approaches that go beyond structural snapshots and ensemble biochemical assays, enabling direct observation of individual transport events.

Single-molecule techniques overcome ensemble averaging and provide high-resolution insights into transport dynamics24–26. In particular, single-molecule Förster resonance energy transfer (smFRET) studies confirmed an alternating-access mechanism in ABC transporters; for example, the bacterial homodimeric exporter McjD requires both substrate and ATP to adopt the OF conformation27. Moreover, combining smFRET with single-particle cryo-EM enabled visualization of the ABC transporter MRP1 under turnover conditions28. Conformational changes in substrate-binding proteins (SBP) from bacterial ABC importers were investigated by smFRET29,30. The SBPs were repurposed as smFRET sensors to track individual translocation events in secondary active transporters, providing quantitative amino acid transport rates31,32. A related sensor derived from the substrate-binding protein OppA from Lactococcus lactis (LlOppA) was also used to characterize substrate binding at the single-molecule level30.

Here, we establish a single-molecule assay that directly visualizes peptide transport by an individual TAP-related heterodimeric ABC transporter. Using an LlOppA-based smFRET sensor encapsulated within liposomes, we monitor substrate translocation by TmrAB at single-molecule resolution. Our results reveal that ATP binding alone is sufficient to induce a conformational switch that drives peptide transport in the absence of Mg2+, whereas Mg2+-dependent ATP hydrolysis is required to reset the transporter and complete the transport cycle.

Results
Establishing an smFRET sensor for antigenic peptides
To detect antigenic peptides of diverse sequences and lengths, similar to those presented on MHC I molecules in adaptive immunity, we exploited the binding promiscuity of a bacterial substrate-binding protein. We used a double-cysteine variant of LlOppA (LlOppAA209C/S441C)30, which was site-specifically labeled at the engineered cysteine residues with the FRET pair Alexa Fluor 555 (AF555) and AF647 (hereafter referred to as OppA) (Supplementary Fig. 1). Binding affinities of the model antigenic peptide RRYQKSTEL to unlabeled and labeled OppA were determined using three independent biophysical approaches: tryptophan fluorescence quenching, ensemble FRET, and single-molecule FRET. All yielded consistent dissociation constants KD ranging from 0.2 to 0.9 µM (Fig. 1d, Supplementary Fig. 1c, e).

To characterize the FRET sensor at the single-molecule level, OppA was immobilized on a PEGylated glass surface using a biotinylated anti-His antibody and streptavidin, with biotin-PEG included at low density for specific tethering. Individual OppA molecules were imaged using total-internal reflection fluorescence (TIRF) microscopy with alternating laser excitation (ALEX) (Fig. 1a, b, Supplementary Fig. 2). In the absence of peptides (apo state), OppA adopted a low-FRET efficiency (E = 0.6), which was shifted to a high-FRET state (E = 0.9) upon addition of increasing peptide concentrations (0–100 µM) (Fig. 1c, Supplementary Figs. 3 and 4). At peptide concentrations near the KD value, OppA exhibited dynamic transitions between low- and high-FRET states, indicating OppA-peptide association and dissociation events (Fig. 1c). Hidden Markov Modeling (HMM) showed concentration-dependent dwell times derived from dynamic smFRET traces (Fig. 1e). The closing rates, ranging from 1.6 to 4.5 s−1, increased linearly with peptide concentrations between 1 and 10 µM (kon = 0.29 ± 0.02 µM−1 s−1 from n = 1311 single-molecule traces) (Fig. 1f), validating the sensor’s capability to monitor peptide concentrations via binding kinetics.

Rationale for single-substrate sensors in liposomes
For single-molecule transport measurements, we employed both wild-type TmrAB (TmrABWT) and a slow-turnover variant generated by substituting the catalytic glutamate with glutamine (TmrAE523QB, hereafter TmrAEQB), which exhibits ~1000-fold reduced ATP hydrolysis13. Both variants were expressed and purified as previously described14 (Supplementary Fig. 5a, b). To track the translocation of individual peptides, liposomes of defined sizes were required to generate lumenal peptide concentrations within the linear detection range of OppA closing rates. Liposomes with a diameter of 100 nm correspond to an internal volume of ~0.5 aL, yielding an effective peptide concentration of ~3.2 µM per translocated peptide. This concentration aligns with the KD value and linear detection range for the closing rates measured by the smFRET sensor (Fig. 1f). To statistically avoid populations of liposomes containing multiple smFRET sensors or transporters, we used a substoichiometric encapsulation/reconstitution ratio of 0.2 OppA and 0.1 TmrAB per liposome (Supplementary Fig. 6a–c).

To selectively capture single uptake-competent transport complexes, we used a conformation-independent nanobody (Nb9F10)14 tethered to the surface via PEG11-biotin and streptavidin (Supplementary Fig. 5c–e). This strategy selectively retained liposomes with correctly oriented, uptake-competent transporters while removing empty liposomes and liposomes with misoriented transporters. Immobilization specificity was confirmed with and without nanobody tethering (Supplementary Fig. 6d). Flow cytometry-based single-liposome assays verified that nanobody binding did not alter transport activity (Supplementary Fig. 7).

Sensor functionality after liposome encapsulation was confirmed by measuring the fluorescence lifetime and anisotropy of the dyes, which showed no membrane-induced perturbations (Supplementary Fig. 8). Functional integrity of the single-substrate sensor was further validated by the addition of the self-inserting nanopore α-hemolysin at saturating peptide concentrations, which triggered a complete transition to the high-FRET state (Supplementary Fig. 6e), demonstrating full sensor functionality inside liposomes.

To validate peptide transport independently of fluorescence, we established a label-free workflow using liquid chromatography-coupled mass spectrometry (LC-MS). LC-MS confirmed ATP-dependent uptake of unlabeled peptides used in single-molecule assays (Supplementary Fig. 9).

Antigenic peptide transport by single transport complexes
Liposomes containing a single TmrABWT were immobilized via a nanobody that captures the transport complex in an uptake-competent orientation. In the absence of Mg-ATP and peptide, the single-molecule sensor OppA encapsulated inside liposomes remained in a low-FRET state (E = 0.6) for ≥80 min at 30 °C (Fig. 2a). Likewise, in the presence of Mg-ADP (3 mM) and peptide (200 µM), no transport occurred over 50 min (Fig. 2b), confirming that Mg-ADP does not support transport and that liposomes are not peptide-leaky. Upon addition of 3 mM Mg-ATP and 200 µM peptide at 30 °C, the sensor gradually shifted to the high-FRET state over several minutes (Fig. 2c), reporting peptide translocation by individual wild-type transporters.

Single-substrate translocation by individual ABC transporters
We next used the slow-turnover variant TmrAEQB, which exhibits a prolonged OF lifetime of ~25 min at 20 °C33, enabling temporal resolution of individual transport events. Neither Mg-ADP nor nucleotide-free conditions resulted in peptide transport (Fig. 3a, b). At 45 °C for 5 min, peptide translocation was observed only in the presence of Mg-ATP (Fig. 3c). After a first transport event, removal of free peptides and nucleotides and imaging at 30 °C allowed the transporter to relay back to the IF state33. A second Mg-ATP/peptide incubation induced a second transport event, evident from an increased high-FRET population (Fig. 3d, e). Control experiments at 30 °C and in the presence of valinomycin excluded effects from a transmembrane potential (Supplementary Fig. 10a–e). A control experiment without Mg-ATP after the first transport event confirmed the absence of liposome leakiness (Supplementary Fig. 10f).

The liposome size distribution immobilized on the microscope surface was assessed by staining with the membrane-associated dye DiD and super-resolution imaging via direct stochastic optical reconstruction microscopy (dSTORM), revealing that most liposomes had a diameter of ~100 nm (Fig. 3g, Supplementary Fig. 11). HMM analysis of smFRET dynamics allowed us to calculate the OppA closing rate and quantify lumenal peptide concentrations of 4 ± 1 µM and 6 ± 1 µM after the first and second translocation events, respectively (Figs. 1f and 3f). These measured lumenal peptide concentrations closely match the theoretical expectations for one and two peptides per 100 nm liposomes (Fig. 3g, Supplementary Fig. 11). For the smFRET measurements, larger liposomes containing multiple FRET sensors were excluded from our analysis. In summary, these data demonstrate that ATP binding alone is sufficient to drive a single peptide-transport event via TmrAEQB.

ATP binding alone without Mg2+ triggers the outward-facing conformation
Adding Mg-ATP to TmrAB induces the transition from the IF to the OF conformation, as shown by previous functional and structural studies14,33. To test whether ATP alone without Mg2+ can drive the conformational switch, wild-type TmrABWT and the slow-turnover variant TmrAEQB were reconstituted into lipid nanodiscs, incubated with ATP-EDTA and the peptide, and analyzed by single-particle cryo-EM (Supplementary Figs. 12 and 13). At 3.02 Å resolution, both TmrABWT and TmrAEQB adopt an outward-facing occluded (OF occluded) conformation (Fig. 4a–c, Supplementary Fig. 14 and Supplementary Table 1). Both structures closely resemble the Mg-ATP-bound OF occluded conformation of TmrAEQB (PDB 6RAI; EMD-4776)14, with root mean square deviations across all Cα atoms of 0.51 Å for TmrAEQB and 0.53 Å for TmrABWT. Two ATP molecules are bound at the canonical and non-canonical nucleotide-binding sites (NBSs), respectively (Fig. 4b, c, Supplementary Fig. 14). Unlike in the Mg-ATP-bound structure14, however, the ATP-EDTA complexes lack Mg2+ coordination: key residues, Q419 and S378 in TmrB, and Q441 and T400 in TmrA, no longer position Mg2+ to coordinate the β- and γ-phosphates of ATP (Fig. 4b, c). A difference EM map comparing TmrAEQB structures in the OF occluded state with Mg-ATP or ATP-EDTA (EMD-4776 vs. EMD-54377) confirms the absence of Mg2+ at both NBSs (Fig. 4d). Notably, no density corresponding to a bound peptide was observed in the cryo-EM map of the OF occluded conformation.

Finally, radiometric ATPase assays performed at 45 °C showed that ATP hydrolysis by wild-type TmrAB (Fig. 4e) and the slow-turnover variant (Fig. 4f) strictly depends on Mg2+. In the presence of 5 mM MgCl2, robust ATP hydrolysis of ~1 s−1 for TmrABWT and ~0.25 min−1 for TmrAEQB was observed, whereas Mg2+ chelation with 10 mM EDTA abolished the ATPase activity (Fig. 4e, f; Supplementary Fig. 15).

ATP binding without Mg2+ drives single-substrate translocation
To clarify the role of Mg2+, we first performed ensemble assays. TmrABWT reconstituted into liposomes showed no detectable transport in the presence of ATP-EDTA, as assessed by both fluorescence-based and LC-MS readouts (Supplementary Fig. 16a, b). In contrast, at the single-molecule level, however, liposomes containing a single reconstituted TmrABWT transporter and pre-incubated with EDTA (10 mM) for 5 min exhibited a single-transport event upon addition of ATP-EDTA and peptide (Supplementary Fig. 16c, e). No additional rounds of transport were detected under these conditions, and multiple turnovers strictly required the presence of Mg2+ (Supplementary Fig. 16d).

Similarly, TmrAEQB liposomes pre-incubated with 10 mM EDTA for 5 min showed no translocation, either in the absence of nucleotide and peptide or when exposed to peptide with ADP-EDTA (Supplementary Fig. 16f, g). Addition of peptide together with ATP-EDTA initiated a transition of OppA to the high-FRET state, indicating successful translocation of a single peptide (Fig. 5a). A second ATP-EDTA addition did not elicit further transport (Fig. 5b), consistent with cryo-EM data showing the transporter locked in the OF state. After an ATP-EDTA-driven translocation event and a 60-min relaxation period, addition of Mg-ATP and peptide restored transporter activity (Fig. 5c, d), confirming that Mg2+ is required to reset the transporter.

Together, these findings demonstrate that ATP binding alone is sufficient to drive a single-substrate translocation event in both TmrABWT and TmrAEQB, while sustained transport cycles strictly require Mg2+-dependent ATP hydrolysis.

Methods
Production and purification of Lactococcus lactis OppA
The double-cysteine variant Lactococcus lactis OppAA209C/S441C (LlOppAAS)30 was expressed in the Escherichia coli MC1061 grown in Terrific Broth (TB) supplemented with 100 µg/mL ampicillin. Cultures were incubated at 37 °C to an OD600 of 0.4, cooled to 25 °C, and induced at an OD600 of 0.6–0.8 with 0.1% (w/v) L-arabinose. Following overnight expression, cells were harvested (4500 × g, 4 °C, 15 min) and stored at −80 °C. All subsequent purification steps were performed at 4 °C. Cell pellets were resuspended in purification buffer (25 mM potassium phosphate (KPi), pH 6.5, 100 mM KCl, 15 mM imidazole, 0.5 mM PMSF, 1 mM DTT) and lysed by sonication (Branson Sonifier 250). The lysate was clarified (18,000 × g, 35 min, 4 °C) and applied to Ni-NTA affinity chromatography (Bio-Rad) pre-equilibrated in equilibration buffer (25 mM KPi, pH 6.5, 100 mM KCl, 15 mM imidazole, 1 mM DTT). After washing with 10 column volumes (CV) of equilibration buffer and 10 CV of wash buffer (25 mM KPi pH 6.5, 100 mM KCl, 30 mM imidazole, 1 mM DTT), the protein was eluted with 8 CV of elution buffer (25 mM KPi pH 6.0, 20 mM KCl, 400 mM imidazole, 1 mM DTT). Further purification was performed by cation exchange chromatography (HiTrap SP HP, Cytiva) with a linear gradient from buffer A (25 mM KPi pH 6.0, 20 mM KCl, 1 mM DTT) to buffer B (25 mM KPi pH 6.0, 500 mM KCl, 1 mM DTT). To remove residual endogenous ligand, LlOppAAS underwent partial unfolding and refolding by stepwise dialysis in decreasing concentrations of guanidine hydrochloride (GuHCl): 2 M (1 h, 8 °C), 1.5 M, 1.0 M, 0.5 M each 30 min, 8 °C. The purified protein was labeled and stored at 4 °C.

Fluorophore labeling of LlOppAAS
Site-specific labeling was performed via maleimide chemistry using Alexa Fluor 555 (AF555) and Alexa Fluor 647 (AF647; Thermo Fisher Scientific). Protein and dyes were mixed at a 1:10:10 molar ratio in OppA-SEC buffer (25 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, and 10% (v/v) glycerol) at 8 °C for 18 h. Excess dyes were quenched with 2 mM β-mercaptoethanol and removed by ZebaTM spin desalting (Thermo Fisher Scientific), followed by size-exclusion chromatography (SEC, Superdex 200 Increase 3.2/300 column, Cytiva) in OppA-SEC buffer. Labeling efficiency was determined from the SEC absorbance at 280 nm, 550 nm, and 650 nm.

Ensemble fluorescence analysis of OppA
Peptide binding of unlabeled or AF555/AF647-labeled OppA was monitored using a Varian Cary Eclipse scanning fluorimeter. Excitation/emission slit widths were set to 5/10 nm. All measurements were performed at room temperature in buffer containing 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5% (v/v) glycerol. For ensemble FRET experiments, OppA (150 nM) was excited at λex = 530 nm, and fluorescence emission spectra were recorded. Tryptophan quenching experiments were performed to probe peptide binding to unlabeled LlOppAAS. Samples were excited at 280 nm, and emission spectra were recorded during titration with increasing peptide concentrations (0.1–10 µM). For titration, OppA was preincubated with either 5 µM or 100 µM RRYQKSTEL.

Production and purification of TmrAB
Recombinant expression and purification of His10-tagged wild-type TmrAB (TmrABWT) and slow-turnover variant TmrAE523QB (TmrAEQB) were performed as described previously9,13,14,33,53. E. coli BL21 (DE3) cells were cultured in LB high-salt medium supplemented with 100 µg/mL ampicillin at 37 °C. Upon reaching an OD600 of 0.6, protein expression was induced with 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG), followed by incubation for 18 h at 20 °C. Cells were harvested by centrifugation (4500 × g, 4 °C, 15 min) and resuspended in lysis buffer (20 mM HEPES-NaOH, pH 7.5, 300 mM NaCl, 0.2 mM PMSF). Cells were lysed by sonication, and debris was removed by centrifugation (18,000 × g, 35 min, 4 °C). Membranes were pelleted (100,000 × g, 2 h, 4 °C), resuspended in lysis buffer, and solubilized for 1 h at 4 °C with 20 mM β-n-dodecyl β-D-maltoside (β-DDM). The solubilized fraction was cleared by ultracentrifugation (100,000 × g, 30 min, 4 °C), and the supernatant was incubated with Ni-NTA agarose resin (Qiagen) for 1 h at 8 °C. After washing with 10 CV of wash buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 1 mM β-DDM, 50 mM imidazole), the protein was eluted with elution buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 1 mM β-DDM, 300 mM imidazole). Eluted fractions were buffer-exchanged into TmrAB-SEC buffer (20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 1 mM β-DDM) using a PD-10 desalting column (Cytiva), followed by size-exclusion chromatography (SEC) on a Superdex 200 Increase 10/300 GL column (Cytiva) in the same buffer. Fractions containing monodisperse TmrAB were pooled, flash-frozen in liquid nitrogen, and stored at −80 °C in TmrAB-SEC buffer supplemented with 20% (v/v) glycerol.

Nanobody production and purification
The nanobody Nb9F10S63C was expressed in E. coli BL21 (DE3) in TB supplemented with 100 µg/mL ampicillin at 37 °C, as described previously14. Expression was induced by the addition of 1 mM IPTG at an OD600 of 0.6, and cells were grown overnight at 28 °C. Cells were harvested by centrifugation (4500 × g, 4 °C, 15 min) and pellets were stored at −80 °C. For purification, pellets were resuspended in nanobody lysis buffer (25 mM HEPES-NaOH, pH 7.4, 300 mM NaCl, 15 mM imidazole, 0.5 mM PMSF) and lysed by sonication. The lysate was clarified by centrifugation (18,000 × g, 35 min, 4 °C) and applied to Ni-NTA resin (Bio-Rad) equilibrated in 25 mM potassium phosphate (KPi) pH 6.5, 100 mM KCl, 20 mM imidazole, and 0.5 mM TCEP. After washing with 10 CV of the same buffer, the bound nanobody was eluted with 8 CV of 25 mM KPi, pH 6.0, 20 mM KCl, 300 mM imidazole, and 0.5 mM TCEP. Eluted fractions were pooled and further purified by cation exchange chromatography using a HiTrap SP HP column (Cytiva) with a linear gradient from low (25 mM KPi pH 6.0, 20 mM KCl, 0.5 mM TCEP) to high salt buffer (25 mM KPi pH 6.0, 500 mM KCl, 0.5 mM TCEP). The nanobody was concentrated and subjected to SEC on a Superdex 200 Increase 10/300 GL column (Cytiva). Site-specific coupling of the nanobody Nb9F10 to biotin-PEG11-maleimide (Sigma-Aldrich) or AF555 (Thermo Fisher Scientific) was performed in the presence of 0.5 mM TCEP at 8 °C for 2 h, using nanobody-to-compound molar ratios of 1:1.2 and 1:5, respectively. Excess linker or fluorophore was removed by ZebaTM spin desalting (Thermo Fisher Scientific), followed by SEC on a Superdex 200 Increase 10/300 GL column (Cytiva).

SDS-PAGE, in-gel fluorescence, and immunoblotting
Protein purity and fluorescence labeling were analyzed by SDS-PAGE. Separation gels (12% (w/v) acrylamide) were prepared with 1.5 M Tris-HCl, pH 8.8, 0.4% (w/v) SDS, 0.05% (w/v) ammonium persulfate (APS), and 0.25% (v/v) N,N,N’,N’-tetramethylethylenediamine (TEMED). Stacking gels (5% (w/v) acrylamide) were prepared with 0.5 M Tris-HCl, pH 6.8, 0.4% (w/v) SDS, 0.1% (w/v) APS, and 0.03% (v/v) TEMED. Gels were used immediately or stored at 4 °C for up to 4 weeks. Protein samples were mixed with 4x Laemmli loading buffer containing dithiothreitol (DTT) and heated to 95 °C for 10 min. Electrophoresis was performed in SDS running buffer (25 mM Tris-HCl, pH 8.3, 192 mM glycine, 0.1% (w/v) SDS) at 180 V. For protein visualization, gels were stained with InstantBlueTM Protein Stain (Expedeon) for 1 h at room temperature with gentle agitation and imaged using the Fusion FX imaging system (Vilber). In-gel fluorescence was recorded using excitation at 555 nm for AF555 (filter set F-595Y3) and 640 nm for AF647 (filter set F-710). Exposure times were optimized individually for each fluorophore.

Proteins separated by SDS-PAGE were transferred to polyvinylidene fluoride (PVDF) membranes by wet electroblotting in phosphate-buffered saline (PBS) pH 8.4 supplemented with 0.1% (w/v) SDS and 20% (v/v) methanol at 30 V for 15 h at 4 °C. Membranes were blocked in PBS supplemented with 0.05% (v/v) Tween-20 (PBS-T) and 5% (w/v) non-fat dry milk for 1 h at room temperature, then incubated with horseradish peroxidase (HRP)-conjugated anti-6xHis (anti-6×His tag® antibody, Abcam, 1:2000 dilution) in blocking solution for 1 h at 4 °C. After three washes with PBS-T, membranes were developed using enhanced chemiluminescence (Clarity Western ECL Substrate, Bio-Rad) and imaged on a Fusion FX imaging system (Vilber).

Liposome preparation
Liposomes were prepared by mixing E. coli polar lipids and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (both from Avanti Polar Lipids) at a molar ratio of 7:3 in chloroform to a final concentration of 5 mg/mL. The lipid mixture was dried under vacuum using a rotary evaporator and subsequently resuspended in liposome buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, and 5% (v/v) glycerol). Large unilamellar vesicles (LUVs) were generated by sonication for 30 min, followed by five freeze-thaw cycles. Liposomes were stored at −80 °C. Before use, LUVs were extruded 11 times through 100 nm polycarbonate membranes using a LiposoFast-Basic extruder (Avestin).

Reconstitution of TmrAB and encapsulation of the single-molecule sensor
TmrABWT and the slow-turnover variant TmrAE523QB (TmrAEQB) were reconstituted into liposomes composed of E. coli polar lipids and DOPC at a molar ratio of 7:3. For single-molecule experiments, a protein-to-lipid ratio (w/w) of 1:10,000 and an OppA-to-liposome ratio of 0.2 molecules per liposome were used. For ensemble transport assays, a protein-to-lipid ratio (w/w) of 1:20 was applied. Extruded LUVs were destabilized by incubation with 0.3% (v/v) Triton X-100 (TX-100) for 30 min. TmrAB and OppA were then added to the detergent-destabilized liposomes and incubated for 30 min at 8 °C under gentle rotation. Detergent removal was performed gradually using sequential additions of polystyrene Bio-Beads SM-2 (Bio-Rad): two incubations at 40 mg/mL (1 h and overnight), followed by two incubations at 80 mg/mL (1 h each). Finally, proteoliposomes were harvested by ultracentrifugation (100,000 × g, 30 min, 4 °C) and resuspended in the appropriate buffer.

Quantification of stochastic TmrAB reconstitution
TmrABWT was labeled with AF647 via maleimide chemistry using an intrinsically occurring single-cysteine residue. Labeling was performed at a 1:10 molar ratio of protein to dye for 2 h at 8 °C on an overhead rotor in TmrAB-SEC buffer. Unreacted fluorophore was removed by SEC in TmrAB-SEC buffer. The labeled TmrAB was reconstituted into liposomes at a protein-to-lipid ratio of 1:10,000 (w/w) as described above. Liposomes containing AF647-labeled TmrABWT were immobilized using a biotinylated PEG11-conjugated nanobody. Data were acquired using a TIRF microscope (NanoImager S, Oxford Nanoimaging). For each region of interest (ROI), 600 frames were recorded with an exposure time of 100 ms. A 640-nm excitation laser was used at 0.9 mW/cm2, and acquisitions were performed in 1-min intervals. Data analysis was carried out using Deep-LASI54. Fluorescence traces were extracted and categorized. Traces in the “bleached” category were further analyzed to identify single- or double-step photobleaching events.

Orientation of TmrAB in liposomes
The membrane orientation of reconstituted TmrAB was determined by proteolytic cleavage of the C-terminal His10-tag on TmrA using Tobacco Etch Virus (TEV) protease. Proteoliposomes (5 mg/mL) were incubated with 0.2 mg/mL TEV protease for 18 h at 8 °C, either in the absence or presence of 1% (v/v) Triton X-100 to differentiate externally versus internally oriented TmrAB populations. Samples were analyzed by SDS-PAGE followed by immunoblotting against the His10-tag to assess protease accessibility.

Peptide synthesis
The 9mer peptides GIINTLEEL, RRYQKSTEL, and RRYCFLKSTEL (FL, fluorescein) were synthesized on pre-loaded Fmoc-L-Leu resin using a Liberty microwave-assisted peptide synthesizer (CEM) following a standard protocol (54 W, 3 min, 75 °C). Each coupling step was performed twice using 0.2 M fluorenylmethoxycarbonyl (Fmoc)-protected amino acid, 0.5 M O-benzotriazole-N,N,N’,N’-tetramethyluronium-hexafluoro-phosphate (HBTU), and 1-hydroxybenzotriazole hydrate (HOBt:H2O). Fmoc deprotection was carried out with 20% (v/v) piperidine and 0.1 M HOBt*H2O in dimethylformamide (DMF). Peptides were cleaved from the resin using a cocktail of 92.5% (v/v) trifluoroacetic acid (TFA), 2.5% (v/v) H2O, 4.5% (v/v) thioanisole, and 0.5% (v/v) 1,2-ethanedithiol (EDT) for 1.5 h at room temperature. The cleaved peptides were precipitated in ice-cold diethyl ether (Et2O), pelleted, dissolved in tert-butanol (tBuOH)/water (4:1, v/v), and lyophilized. Peptides were purified by reverse-phase (RP)-C18 HPLC (PerfectSil C18 column; MZ-Analysentechnik) using buffer A (Milli-Q water with 0.05% (v/v) TFA) and buffer B (acetonitrile with 0.05% (v/v) TFA). Peptide identity and purity were confirmed by LC-MS. For fluorescence labeling, the peptide RRYCKSTEL was dissolved in PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), supplemented with 3.5 mM DMF and incubated for 1 h at room temperature with a 1.3-fold molar excess of 5-iodoacetamidofluorescein (Merck).

Mass spectrometry
All recombinantly produced proteins and chemically synthesized peptides were analyzed by liquid chromatography-coupled mass spectrometry (LC-MS) using a Waters BioAccord system operating with UNIFY 3.1.0 software. Peptides were separated on an Acquity BEH C18 column (1.7 µm, 2.1 × 100 mm) using a 6.5-min linear gradient from 2 to 80% acetonitrile in Milli-Q water. Proteins were separated on an Acquity BEH C4 column (1.7 µm, 2.1 × 50 mm) using a 6.5 min linear gradient from 5 to 80% acetonitrile in Milli-Q water; both mobile phases were supplemented with 0.1% formic acid. Spectra were acquired in positive ion mode with a cone voltage of 30 V and a capillary voltage of 0.8 kV.

LC-MS-based peptide transport assay
Peptide translocation by TmrABWT was measured using LC-MS. Proteoliposomes (2 mg/mL final) were incubated at 45 °C for 45 min with RRYQKSTEL (50 µM or 200 µM), GIINTLEEL (50 µM), or the fluorescein-labeled peptide RRYCFLKSTEL (C4F, 5 µM) in the presence of 3 mM MgCl2 and 3 mM ATP or ADP. All reactions contained an ATP regeneration system (0.5 mg/mL creatin kinase and 10 mM creatine phosphate) in liposome buffer. Reactions were stopped with ice-cold liposome buffer containing 10 mM EDTA. Samples were ultracentrifuged (450,000 × g, 30 min, 4 °C), and pellets were resuspended in 200 µL of 100 mM Na2CO3 (pH 11.5), followed by a second ultracentrifugation under the same conditions. Final pellets were resuspended in Milli-Q water and sonicated for 2 min. For quantification, C4F was spiked into samples containing RRYQKSTEL and GIINTLEEL as an internal standard. A final ultracentrifugation (450,000 × g, 1.5 h, 4 °C) was performed before LC-MS analysis using a Waters BioAccord system (UNIFY 3.1.0). Peptides were separated on an Acquity BEH C18 column (1.7 µm, 2.1 × 50 mm) and analyzed in positive ion mode using a cone voltage of 30 V and capillary voltage of 0.8 kV. Quantification was based on total ion chromatogram peak areas at the peptide-specific retention times.

Filter plate-based peptide transport assay
Peptide transport by TmrABWT was quantified using the fluorescent peptide RRYCFLKSTEL (C4F) in a 96-well filter plate assay. Proteoliposomes (1 mg/mL final) were incubated at 45 °C for 15 min with varying concentrations of C4F, 3 mM MgCl2, and 3 mM ATP or ADP in liposome buffer. Transport was terminated by the addition of ice-cold liposome buffer containing 10 mM EDTA. Samples were transferred to polyethyleneimine (PEI)-precoated 96-well filter plates (Durapore membrane, 0.65 µm pore size, Millipore) and washed five times with 200 µL stop buffer to remove free peptides. Encapsulated C4F was released by incubating wells with PBS (pH 7.4) containing 0.1% (w/v) SDS for 10 min. The resulting lysates were transferred to black 96-well microtiter plates, and fluorescence was measured using a CLARIOstar plate reader (BMG LABTECH, Clariostar 5.20 R5, Mars 3.10 R5) at excitation/emission wavelengths of 485/520 nm.

Single-liposome transport monitored by flow cytometry
Single-liposome transport was quantified using a flow cytometry-based assay as previously described33. Proteoliposomes containing TmrABWT were labeled with AF555-conjugated nanobody (Nb9F10S63C-AF555) at a TmrAB/nanobody molar ratio of 1:2. Labeled proteoliposomes were washed twice by ultracentrifugation (270,000 × g, 30 min, 4 °C) and incubated with 30 µM C4F peptide, 5 mM MgCl2, 3 mM ATP or ADP for 10 min at 45 °C. Following incubation, samples were washed twice using the same ultracentrifugation conditions to remove unbound peptide. Mean fluorescence intensities corresponding to nanobody-tagged TmrAB and transported C4F peptide were measured using a FACS Celesta flow cytometer (BD Biosciences, BD FACSDiva 8.0.1.1). A total of 105 proteoliposomes were recorded per sample based on forward scatter (FSC) and side scatter parameters. Single-liposome events were gated by FSC height versus area, and AF555-positive events were further gated to selectively analyze nanobody-decorated proteoliposomes. Data were processed using FlowJo software (v10.6.1). All measurements were performed in triplicates, and results are presented as mean ± s.d.

Fluorescence anisotropy
Fluorescence anisotropy measurements were performed to assess the dynamics of AF555- and AF647-labeled OppA. Experiments were conducted in buffer containing 25 mM HEPES-NaOH pH 7.5, 150 mM NaCl. Labeled OppA was mixed with liposomes at a 1:1 (w/w) protein-to-liposome ratio. Measurements were performed using a CLARIOstar plate reader (BMG LABTECH) with excitation/emission settings of 482/535 nm and a long-pass dichroic filter LP504.

Time-correlated single-photon counting (TCSPC)
Fluorescence lifetime measurements were performed using a FluoTime 100 spectrometer (PicoQuant) equipped for time-correlated single-photon counting. Experiments were conducted in 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, and included free dye, OppA, and liposome-encapsulated OppA samples. AF555 and AF647 were excited at 510 nm and 610 nm, respectively. Emission was detected through a 620/60 nm bandpass filter for AF555 and a BG470 0 nm long-pass filter for AF647. Photon arrival times were recorded until the TCSPC histogram reached a peak of 10,000 photons. Fluorescence decay curves were analyzed by fitting mono- or biexponential decay models using FluoFit (PicoQuant).

Functionalization of glass slides for single-molecule FRET analysis
Glass coverslides were functionalized via aminosilanization as previously described55. Briefly, coverslides were cleaned by sequential sonication in Milli-Q water and analytical-grade acetone (>99.9%), followed by oxygen plasma treatment (0.3 mbar, 80% power, 15 min), and a 10 min incubation in methanol (100 mL). Subsequently, coverslides were incubated for 20 min in silanization solution containing 100 mL methanol (analytical grade), 5 mL acetic acid, and 3 mL 3-aminopropyltrimethoxysilane (APTES). After brief sonication (1 min), the slides were incubated for an additional 10 min, rinsed four times with methanol, and dried under a stream of nitrogen gas. To functionalize the surface with polyethylene glycol (PEG), a mixture of biotinylated-PEG (4% v/v) and non-biotinylated PEG was coupled to the amino groups via N-hydroxysuccinimide (NHS) chemistry. Specifically, 3.5 mg of biotin-PEG-NHS (5 kDa) were dissolved in 750 µL of 0.1 M NaHCO3 PEGylation-buffer (pH 8.5), followed by the addition of 85 mg CH3O-PEG-NH-CO-C3H6-CONHS (5 kDa). Air bubbles were removed by centrifugation at 16,000 × g for 1 min. The PEG solution was sandwiched between two coverslips and incubated overnight in a humidity chamber. Coverslides were then rinsed thoroughly with Milli-Q water, dried with nitrogen gas, and subject to a second PEGylation step using CH3-PEG-NHS (333 Da) in PEGylation-buffer (25 mM final) in a similar sandwich configuration. This second PEGylation was performed overnight under humid conditions. Finally, slides were rinsed with Milli-Q water, dried under nitrogen gas, and stored at −20 °C under argon gas until use.

Liposome size distribution on the microscope surface
Liposomes containing single TmrABWT were stained with the lipophilic carbocyanine dye DiD (0.1 mol%) and tethered to the glass surface via biotinylated PEG11-conjugated nanobody Nb9F10. Fluorescence intensities were quantified by dSTORM, integrating the signal within a circular region of constant area surrounding each liposome. The resulting integrated fluorescence intensity per area was converted into liposome diameters by calibrating the NanoImager S (Oxford Nanoimaging) scale bar against TetraSpeckTM calibration beads (Thermo Fisher Scientific). The fluorescence signal from a 100 nm liposome was used as a reference to convert the fluorescence intensities into liposome diameters.

Theoretical calculation of a single peptide encapsulated inside liposomes
The concentration corresponding to a single peptide encapsulated in a spherical liposome was calculated as c = n/V, where n = 1/NA (Avogadro constant) and V = 4/3 πr3 is the liposome volume.

Single-molecule FRET imaging
Single-molecule FRET experiments were conducted using a flow chamber system to immobilize liposomes and transporters in an uptake-competent orientation. Flow chambers were assembled by placing a biotin-PEG-functionalized glass slide (4% (v/v) biotin-PEG) onto a sticky-slide flow channel (ILuer 0.8 mm, Ibidi), with the functionalized surface facing inward. The flow chamber was connected via silicon tubing, and flow was controlled using a 20 mL syringe. The chamber was flushed with 1 mL liposome buffer, followed by incubation with streptavidin (0.2 mg/mL) for 30 min at 8 °C to enable binding to the biotin-PEG surface. After washing with 1 mL liposome buffer, the surface was functionalized by incubating with either biotinylated anti-His antibody (0.02 mg/mL) (Abcam) or biotinylated-PEG11-Nb9F10S63C (0.3 mg/mL) for 30 min at 4 °C, followed by an additional rinse with 1 mL liposome buffer. For anti-His antibody tethering, 100 nM fluorophore-labeled OppA was introduced into the flow chamber. For nanobody-based tethering, proteoliposomes (5 mg/mL) were centrifuged at 2000 × g for 2 min at 4 °C and subsequently incubated on the surface for 30 min at 8 °C. Unbound material was removed by five successive washes with 1 mL liposome buffer, followed by 1 mL imaging buffer (25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5% (v/v) glycerol, 90 mM glucose, 5 mM Trolox, pyranose oxidase (7.5 U/mL), and catalase (1 kU/mL)). For the experiments involving valinomycin, 10 mM KCl was added to the imaging buffer. For single-translocation events with TmrAEQB, proteoliposomes were incubated with pre-heated imaging buffer at 45 °C for 5 min followed by incubation with 200 µM RRYQKSTEL peptide and 3 mM ATP in pre-heated imaging buffer at 45 °C for 5 min. Subsequently, excess peptide and ATP were removed by washing with 1 mL cold liposome buffer and 0.7 mL imaging buffer. The second translocation event was initiated by following the same procedure. For ATP-EDTA experiments, liposomes were pre-incubated with 10 mM EDTA for 5 min to chelate divalent cations. Imaging was performed using alternating laser excitation (ALEX) on a TIRF microscope (NanoImager S, Oxford Nanoimaging). Typically, 600 frames were acquired per ROI using an exposure time of 100 ms. Laser powers were set to 0.8 mW/cm2 (532 nm excitation) and 0.9 mW/cm2 (640 nm excitation), with data recorded in 1-min intervals.

Single-molecule FRET data analysis
Single-molecule FRET (smFRET) measurements were performed using ALEX, allowing the assignment of detected photons based on both excitation and emission wavelength. Photon counts were classified into three detection channels: donor excitation with donor emission (FDD), donor excitation with acceptor emission (FDA), and acceptor excitation with acceptor emission (FAA). From these signals, FRET efficiency (E) and stoichiometry (S) were calculated and FRET traces extracted using NanoImager software (ONI Nanoimager Development Build) and analyzed with deepFRET56. An 80% confidence threshold was applied for automated trace selection. Following automated classification, traces were manually curated to ensure data quality. Dynamics smFRET traces were further analyzed via HMM by deepFRET. One-dimensional histograms of E and S values were generated and visualized using OriginPro 2024 (OriginLab) and fitted with Gaussian distributions to identify distinct FRET populations.

ATPase activity
ATP hydrolysis by TmrAEQB and TmrABWT was quantified using radiolabeled ATP. TmrAB reconstituted into liposomes (0.2 mg/mL) at a protein-to-lipid ratio of 1:20 (w/w) were incubated with 2 mM ouabain, 10 mM NaN3, 50 µM EGTA, and 0.3 mM ATP spiked with [γ32P]ATP (Hartmann Analytic), supplemented with either 5 mM MgCl2 or 10 mM EDTA. Reactions were incubated for 18 min at 45 °C with or without 200 µM RRYQKSTEL peptide. Non-specific background hydrolysis was assessed in parallel for reactions lacking TmrAB. After incubation, aliquots were spotted onto polyethyleneimine cellulose thin-layer chromatography (TLC) plates (Merck Millipore). TLC separation was performed using 0.8 M LiCl in 0.8 M acetic acid as the mobile phase. Plates were dried, exposed overnight in an Exposure Cassette-K (Bio-Rad), and radiolabeled products were visualized using a Personal Molecular Imager System (Bio-Rad).

Reconstitution in lipid nanodiscs
Reconstitution of wild-type TmrAB (TmrABWT) and the slow-turnover variant TmrAEQB into lipid nanodiscs was performed as previously described14. Briefly, bovine brain lipids (Sigma-Aldrich) were solubilized in 50 mM HEPES, pH 7.4, 150 mM NaCl supplemented with 20 mM β-DDM at 37 °C for 30 min. Purified TmrAB, MSP1D1 scaffold protein, and solubilized lipids were mixed at a molar ratio of 1:7.5:100 (TmrAB:MSP1D1:lipid) in TmrAB-SEC buffer lacking detergent. The final lipid-to-detergent ratio was 1:1.4 (w/w). The mixture was incubated at 20 °C for 30 min before initiating nanodisc self-assembly by sequential addition of Bio-Beads SM-2 (Bio-Rad) at 8 °C: 40 mg/mL for 1 h, followed by 80 mg/mL overnight to remove detergent. Following detergent removal, nanodisc-containing samples were concentrated using Amicon Ultra centrifugal filters (0.5 mL, 100 kDa MWCO; Merck Millipore). To label the complexes, reconstituted TmrAB-nanodisc complexes were incubated with the nanobody Nb9F10S63C at a 1:1 molar ratio for 10 min at 20 °C. Subsequently, samples were analyzed by size-exclusion chromatography (SEC) using a Superdex 200 Increase 3.2/300 column (Cytiva) equilibrated in TmrAB-SEC buffer without detergent to separate nanobody-bound TmrAB containing nanodiscs from empty nanodiscs.

Cryo-EM sample preparation and data acquisition
Nanodiscs-reconstituted TmrABWT and TmrAEQB bound to nanobody were incubated with 10 mM ATP, 10 mM EDTA, and 200 µM peptide RRYQKSTEL for 5 min at 45 °C. Immediately thereafter, 3 µL of each sample was applied to freshly glow-discharged Quantifoil grids (R1.2/1.3, 300 mesh), blotted, and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific) operated at 4 °C and 100% humidity. Cryo-EM data were collected on a 200 kV Glacios transmission electron microscope (Thermo Fisher Scientific, EPU 3.10.0) equipped with a Falcon 3EC direct electron detector (Thermo Fisher Scientific) operated in counting mode. Micrographs were acquired at a nominal magnification of 150,000×, corresponding to a calibrated pixel size of 0.95 Å. Dose-fractionated movies were recorded at an electron flux of 0.8 e− pixel−1 s−1 over a total exposure time of 33.35 s distributed over 24 frames, resulting in a cumulative dose of 28.3 e− Å−2 (see Supplementary Table 1).

Cryo-EM image processing
Cryo-EM data were processed using cryoSPARC v4.6.0–4.7.0 (ref. 57), following a common workflow for both TmrABWT and TmrAEQB, as illustrated in Supplementary Figs. 12 and 13. After Patch Motion Correction and Patch CTF estimation, micrographs were curated by selecting those with CTF fits better than 5 Å for downstream processing. Particle picking was performed using TOPAZ 0.2.5a (ref. 58), followed by particle extraction with three-fold Fourier cropping (2.85 Å pixel−1). Extracted particles were subjected to multi-class ab initio reconstruction, and classes displaying a feature characteristic of type IV ABC transporters were selected for iterative heterogeneous refinement to enrich for high-quality TmrAB particles. Selected particles were then re-extracted at full pixel size (0.95 Å pixel−1) and subjected to non-uniform refinement, yielding maps at 3.26 and 3.20 Å resolution for TmrABWT and TmrAEQB, respectively, based on the gold-standard Fourier shell correlation (FSC = 0.143) criterion. To further improve resolution, reference-based motion correction and CTF refinement were applied, followed by a final round of non-uniform refinement. This resulted in final reconstructions at 3.02 Å resolution for both TmrABWT and TmrAEQB.

Molecular modeling
The previously determined structure of TmrAEQB in OF occluded conformation (PDB: 6RAI) was used as a starting model and docked into the corresponding cryo-EM maps using UCSF ChimeraX 1.9 (ref. 59). For modeling TmrABWT, residue 523 was reverted from glutamine to the native glutamic acid. Both TmrABWT and TmrAEQB models were independently refined in real space against their respective cryo-EM density maps using Coot 0.9.8.95 (ref. 60) for manual model adjustment and Phenix 1.21.2-5419 (ref. 61) for automated refinement.

Statistical analysis
Statistical significance analysis was performed using GraphPad Prism 8.0.2. ATPase data were analyzed by two-way ANOVA. Statistical tests and P values are reported in the figure legends. For TmrABWT, the F value for the comparison of MgCl2 versus EDTA without the peptide is 337.7, and with the peptide, 338.8, with a degree of freedom of dfbetween = 1 and dfwithin = 4. For TmrAEQB, the F value for the comparison of MgCl2 versus EDTA without the peptide is 268, and with the peptide, 87.7, with a degree of freedom of dfbetween = 1 and dfwithin = 4.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "see pdf - Recommendatio is to work on and resubmit the paper with major revisions.", "reviewer_2": "The manuscript by Nocker is probing the role of Mg2+ in either coordinating ATP or facilitating hydrolysis and subsequent substrate transport of the ABC transporter TmrAB. The authors use a smFRET approach of TmrAB reconstituted in liposomes to monitor peptide transport by utilising the OppA as reporter. They evaluated their system in the presence of ATP-Mg2+ and peptide, with wt or ATPase deficient TmrAB EQ mutant. They also pretreated their proteoliposomes with EDTA and added ATP-EDTA, and they observed that a single peptide transport event takes place. Based on these observations they conclude that ATP alone is capable to drive transport but not resetting the transporter for a subsequent transport event. Finally, using cryo-EM analysis of TmrAB with ATP-EDTA, they show that the transporter is capable of adopting the outward facing conformation without Mg2+ further validating their findings.\nThe work is built on the authors expertise to study TmrAB. The experiments have been executed to high standards and with fairly sound conclusions. Some of the results such as the transport of single peptide is just a reconfirmation of their previous work about TmrAB transporting a single peptide per transport event. Debugging the role of Mg2+ is the novelty of this study.\nMy main concern is the lack of discussion on the presence of OppA species that do not show high FRET upon a transport event in all their experiments; under all conditions with ATP and peptide, the plots show a bimodal behaviour that the authors have not addressed and it presents a limitation of using OppA as the reporter rather than labelling TmrAB directly. This is the biggest weakness of this manuscript. The plots show a significant portion of OppA molecules not binding the peptide even under conditions of saturation. This could either be due to the presence of more than one TmrAB in the liposome (there is no evidence for this) or encapsulation of more than one OppA or a more complex transport kinetic model that needs further investigation.\nThe legend for Fig 3 f on ‘transition kinetics’ are not relevant to TmrAB as the FRET is only reporting on OppA binding kinetics which is not the focus of this study. This should be changed.\nFig2 would also benefit from having a HMM analysis table as Fig3, and make comparisons between the wt and mutant protein in the discussion.\nUnder the cryo-EM results section, the manuscript would benefit by providing a comparison of the NBDs with and without Mg2+; ie do the side chains adopt different rotamers? They should also provide rmsd values for the two structures. Are there any conformational changes within the NBD/coupling helix region that the absence of Mg2+ can induce?\nThe statement in page 10 ‘Yet our work uncovers a previously overlooked Mg2+- independent ATP-bound state that supports single-turnover transport.’ is confused with their previous sentence and there is not much relevancy for transporters which are greatly uncoupled for ATP-dependent substrate transport.\nSupplementary Table 1 is reporting the EQ mutant structure but there is no figure of its ATP binding site as for the wt and any discussion beyond the difference maps. Supplementary Fig 8 should show the process of both wt and EQ mutant separately.", "reviewer_3": "Single-molecule dynamics reveal ATP binding alone powers substrate translocation by an ABC transporter.\nThe authors show that binding of ATP is sufficient for a half turnover of the peptide ABC transporter TmrAB, i.e. the transport of a peptide from cis to trans. Mg-ATP is required for hydrolysis and the other half turnover, which resets the system for the next translocation event. Thus, the conformational switch from inward-facing to outward-facing by ATP (without Mg2+ ions) is a key finding of this paper, which has been postulated before but may not have demonstrated as convincingly as done for TmrAB in this paper. Perhaps, the authors should summarize in their discussion to what extent the ATP-driven half turnover is unique for TmrAB (or heterodimeric ABCs), and indicate other studies where this has been shown directly or indirectly or ruled out. Now only computational studies and CFTR (special case) are mentioned in the Discussion section.\nOverall, this is a beautifully executed single-molecule study on an important bacterial homologue of the human TAP system. The work is important and well presented. We have a few questions and minor comments for the authors to consider:\n1. Figure 1d reports Kd=0.9 uM (text indicates values from 0.2-0.9 uM) for RRYQKSTEL binding to OppA; Figure 1f reports kon and tau, from which the Kd can be calculated, which then yields Kd=7uM. What is the explanation for the approximately 10-fold difference in binding constant?\n2. The internal volume of the vesicles is calculated for a diameter of 160 nm, whereas the peak in the vesicle concentration is around 200 (Fig. 3g). Why is 160 nm chosen, and how does the distribution of vesicles sizes (from 100 to 500 nm) affect the analysis of the data (e.g. in the context of max 1 OppA and 1 TmrAB per vesicle, and both proteins need to be in the same vesicle)?\n3. SFig.4 shows that nanobody binding does not affect the activity of TmrAB. Since a high protein-to-lipid ratio is used, what is the evidence that the reported activity is actually transport and not peptide binding? The same applies to the data of SFig.6. It should be possible to discriminate binding from transport by comparing the number of peptides translocated with the number of TmrAB molecules in the membrane.\n4. Do non-hydrolyzable ATP analogues also trigger the half turnover of TmrAB?\n5. How was OppA encapsulated in the vesicle lumen? Page 23 only describes the membrane reconstitution of TmrAB.\n6. Figure 2ab show a high FRET peak, which is not seen in 3ab. Is this endogenously bound peptide in this particular preparation of OppA?\n7. Figure 2c shows the time dependence of peptide translocation (0-10 and 11-20 min) for wildtype TmrAB, but this is not shown for the EQ mutant in Figure 3c or 3d. Shouldn’t it be an order of magnitude slower?\n8. Figure 6 suggests that 1 ATP is hydrolyzed per peptide translocated. Is there evidence for this contention; they also write on page 10 that wildtype TmrAB exhibits futile hydrolysis of ATP.\n9. Page 28: It is not entirely clear how FRET traces were selected for the FRET distributions. Were traces without dynamics discarded? What were the criteria?\n10. SFig. 3ab: Why a double peak in the SEC profile for wildtype TmrAB, which is not seen for the EQ protein.\n11. SFig. 7a: The high FRET state after one translocation event is similar to the high FRET state after two translocation events of Fig. 3d. Similarly, in SFig. 10e, the high/low FRET ratio is lower after translocation event 2 than after 1 (and even after 3 events it may still be lower than after 1). A brief description on variations between experiments and reproducibility or other explanation would be helpful.\n12. The description of the legend of SFig. 10g (Hidden Markov Model) is ambiguous and needs some rewriting.", "reviewer_4": "ABC transporters represent a large family of primary active membrane transporters, which are conserved across all domains of life. They utilize ATP binding and hydrolysis energy to transport diverse substrates against concentration gradients across cellular membranes. Their involvement in human diseases, including multidrug resistance and genetic disorders, makes understanding their molecular mechanisms relevant. ABC proteins are categorized based on their nucleotide-binding domains (NBDs) and transmembrane domains (TMDs). While structural studies provided detailed insights into structural aspects of the transport mechanisms, single-molecule techniques have begun to overcome limitations of ensemble methods, providing insights into transport dynamics and revealing novel mechanistic details of substrate translocation events. In the submitted paper, a novel single-molecule platform combining FRET-based peptide sensing with conformationally selective nanobodies aims at direct observation of individual ABC transporter substrate translocation events.\nThe studied system was TmrAB a heterodimeric ABC transporter that mediates peptide translocation across bacterial membranes, functioning as a structural and functional homolog of the human TAP complex. Using a slow-turnover TmrAB variant (TmrAEQB), the authors claim to observe discrete transport events at the single transporter level. Their interpretation is that ATP binding alone, even without Mg2+, can drive substrate translocation through an IF-to-OF conformational switch. These findings support a mechanistic model where ATP binding induces NBD dimerization and conformational switching, with peptide translocation occurring concurrently, followed by Mg2+-dependent ATP hydrolysis for transporter resetting. The authors suggest that their work establishes a single-molecule framework for understanding ABC transporter mechanisms, though limitations include temperature constraints for wild-type TmrAB studies and sensor saturation after few uptake events.\nI find the topic and the approach of the paper timely, innovative, and very interesting, and in conclusion worthwhile to publish. The study was in parts done carefully, yet various conclusions and the main claim of the paper are not supported by the supplied data and the modelling. I thus render the paper in principle suitable for publication in Nature Communications due to its novelty and ideas, subject to the following major revisions that require additional experiments/data and analysis:"}, "author_response": "Point-to-Point Response to Reviewers (NCOMMS-25-68980-T):\nReviewer #1 (Remarks to the Author)\nABC transporters represent a large family of primary active membrane transporters, which are\nconserved across all domains of life. They utilize ATP binding and hydrolysis energy to transport diverse\nsubstrates against concentration gradients across cellular membranes. Their involvement in human\ndiseases, including multidrug resistance and genetic disorders, makes understanding their molecular\nmechanisms relevant. ABC proteins are categorized based on their nucleotide-binding domains (NBDs)\nand transmembrane domains (TMDs). While structural studies provided detailed insights into\nstructural aspects of the transport mechanisms, single-molecule techniques have begun to overcome\nlimitations of ensemble methods, providing insights into transport dynamics and revealing novel\nmechanistic details of substrate translocation events. In the submitted paper, a novel single-molecule\nplatform combining FRET-based peptide sensing with conformationally selective nanobodies aims at\ndirect observation of individual ABC transporter substrate translocation events.\nThe studied system was TmrAB a heterodimeric ABC transporter that mediates peptide translocation\nacross bacterial membranes, functioning as a structural and functional homolog of the human TAP\ncomplex. Using a slow-turnover TmrAB variant (TmrAEQB), the authors claim to observe discrete\ntransport events at the single transporter level. Their interpretation is that ATP binding alone, even\nwithout Mg2+, can drive substrate translocation through an IF-to-OF conformational switch. These\nfindings support a mechanistic model where ATP binding induces NBD dimerization and\nconformational switching, with peptide translocation occurring concurrently, followed by Mg2+-\ndependent ATP hydrolysis for transporter resetting. The authors suggest that their work establishes a\nsingle-molecule framework for understanding ABC transporter mechanisms, though limitations\ninclude temperature constraints for wild-type TmrAB studies and sensor saturation after few uptake\nevents.\nI find the topic and the approach of the paper timely, innovative, and very interesting, and in conclusion\nworthwhile to publish. The study was in parts done carefully, yet various conclusions and the main\nclaim of the paper are not supported by the supplied data and the modelling. I thus render the paper\nin principle suitable for publication in Nature Communications due to its novelty and ideas, subject to\nthe following major revisions that require additional experiments/data and analysis:\nReply: We sincerely thank Reviewer #1 for the thoughtful and thorough evaluation of our manuscript.\nWe are very grateful for the positive feedback regarding the timeliness, innovation, and overall\ninterest of our study. We are encouraged by the assessment that our work is, in principle, suitable for\npublication in Nature Communications due to its novelty and conceptual contribution. We also\nappreciate the constructive feedback pointing out areas where additional experiments, data, and\nanalysis are needed. We have carefully addressed all concerns raised, which we believe considerably\nstrengthen both the robustness and clarity of our mechanistic conclusions.\nSpecific Comments:\n1) The paper lacks a summary of smFRET work on ABC transporters from various groups in which\nthe major mechanistic conclusions should be summarized and the advances made (also in\ncomparison to the current approach) are compared in a fair way\nReply: We thank the reviewer for this important suggestion. We have now expanded the\nintroduction to include a concise summary of previous smFRET studies on ABC transporter and\ntheir mechanistic insights. This addition also clarifies how our approach complements and\nadvances beyond these earlier works. The New Paragraph (page 3) reads:\n“In particular, single-molecule Förster resonance energy transfer (smFRET) studies confirmed\nan alternating-access mechanisms in ABC transporters; for example, the bacterial homodimeric\nexporter McjD requires both substrate and ATP to adopt the outward-facing conformation\n1\n(Husada et al, 2018). Moreover, combining smFRET with single-particle cryo-EM has enabled\nvisualization of the ABC transporter MRP1 under turnover conditions (Wang et al, 2020).\nConformational changes in substrate-binding proteins (SBP) from bacterial ABC importers were\ninvestigated by smFRET (de Boer et al, 2019; Husada et al, 2015). The SBPs were repurposed as\nsmFRET sensors to track individual translocation events in secondary active transporters,\nproviding quantitative amino acid transport rates (Ciftci et al, 2020; Fitzgerald et al, 2019).“\nThis addition summarizes prior single-molecule approaches, acknowledges their contributions,\nand positions our work within the broader context of smFRET-based ABC transporter research.\n2) Relevant references in which single-transporter assays were introduced are not mentioned\n(Stamou group and others); these should be integrated into the introduction and the framing\nof the paper.\nReply: We appreciate the reviewer pointing out this omission. Although the Stamou’s work was\nalready included in the original manuscript, it was not cited in the introduction. We now\nreference these key methodological contributions directly in the introduction:\n“Single-molecule techniques overcome ensemble averaging and provide high-resolution\ninsights into transport dynamics (Blanco & Walter, 2010; Holden et al, 2010; Kosmidis et al,\n2022).\nThe work is also discussed in the Discussion (Kosmidis et al, 2022), where we compare it to the\n“resting” state observed in our system. This strengthens the framing and situates our study\nwithin the broader landscape of single-transporter methods.\n3) The capabilities of the developed assay should be compared against other format (current ref.\n26-27), which allow real-time recordings. What are the pros and cons of the approach\nintroduced?\nReply: We thank the reviewer for this useful suggestion. We have expanded the Discussion to\nexplicitly compare our assays with real-time assay formats (Ciftci et al, 2020; Fitzgerald et al,\n2019), highlighting advantages and limitations.\nIn brief, our assay offers several advantages:\n(i) Selective immobilization of uptake-competent transporters via a conformationally non-\nselective nanobody.\n(ii) Direct observation of peptide translocation through an ABC transporter at the single-\nmolecule level.\nThese features allow mechanistic dissection of transport steps that remain hidden in ensemble\nassays.\nThe main limitations, now stated more explicitly, include:\n(i) Inability to perform single-molecule assays on wild-type TmrAB under fully\nphysiological conditions (>45 °C) due to instrument and sensor thermostability\nconstraints.\n(ii) Sensor saturation after a few uptake events, which currently precludes determining\nwhether transport cycles proceed in bursts or continuously.\nThis expanded discussion highlights the complementary nature of our assays relative to existing\nreal-time formats and provides a balanced view of its strengths and constraints.\n2\n4) Figure 1; please add supplementary data of traces of all conditions with at least 20-30 traces\nper condition.\nReply: We thank the reviewer for this suggestion. We have now added representative single-\nmolecule FRET traces for all conditions in the New Supplementary Figs. 3 and 4, providing\ncomprehensive visualization of the single-molecule behavior across all experimental conditions.\n5) Figure 1d; analysis of data is not correct or not correctly described. Normalized acceptor\nfluorescence is not identical to the area of the “blue” closed-state population shown in panels\nc; it would have to be the normalized area of one against the other population. According to\npanel c, there is saturation at closed-state populations of 60% so the panel needs attention\nReply: We appreciate the reviewer’s accurate observation. We have corrected the legend and\ntext associated with Fig. 1d to clearly distinguish between normalized acceptor fluorescence\nand the population areas derived from Gaussian fits in Fig. 1c. This removes the previous\nambiguity.\n6) Figure 1c; please explain why the sensor has the limitation that only 60% are active or why the\nsensor does not saturate in the fully closed state? The problem persists in liposomes\n(Figure S2) but here also the apo state shows a substantial proportion of closed state in the\nabsence of peptide. Maybe analysis of traces from individual liposomes can help? Please\ncomment and explain.\nReply: We thank the reviewer for raising this important point. The apparent plateau at ~60%\ndoes not reflect intrinsic limitation of the sensor. Instead, it arises from constraints in Gaussian\ndeconvolution when fitting small subpopulations in FRET histograms. As a result, the\nhigh-FRET fraction is underestimated, whereas inspection of the raw, higher-resolution\nhistogram shows a larger proportion of molecules in the closed/high-FRET state. A similar\nunderestimation was also observed in our initial liposome measurements. We have now clarified this in the\nmanuscript and added a brief explanation in the figure legend.\nAs outlined in our response to Reviewer #2 (comment 1), the histograms represent a mixture of\nlow- and high-FRET OppA populations. Peptides are released after 0.5 s resulting in a shift from\nhigh to low FRET and the peptide concentration determines how fast the reassociation happens.\nA small low FRET population will be always present and achieving complete sensor saturation\nrequires multiple peptide translocation events and very high peptide concentrations. To directly\ndemonstrate this, we performed an additional experiment in which three success translocation\nevents mediated by TmrAEQB resulted in full sensor saturation (Fig. R2).\nImportantly, after optimizing OppA purification and sample handling, we were able to eliminate\nnearly all of the high-FRET apo population. The New Supplementary Fig. 17 (AMP-PNP control)\nconfirms a clean low-FRET baseline in the absence of peptide.\nFinally, we note that our HMM analysis is based solely on dynamic, single-liposome trajectories\nand is not affected by these histogram-fitting artifacts.\n7) Figure 1e,f; please provide the dwell-time histograms of the different conditions used to\ncalculate rates in panel f; to me the trace shown in e, which I assume is close to K , should have\nD\na closed-state lifetime longer than 500 ms; more data/analysis is needed to give confidence in\nthe analysis\nReply: We thank the reviewer for emphasizing the importance of dwell-time analyses. As noted,\nindividual OppA molecules exhibit considerable heterogeneity in closed-state lifetimes, even\nwithin the same smFRET trajectory. Some closing events last only a few milliseconds, while\nothers persist for seconds. This variability reflects the intrinsic stochastic dynamics of OppA and\nillustrates why single-molecule measurements are essential to resolve behaviors that are\naveraged out in ensemble analyses.\nThe rates reported in Fig. 1f are based on a large dataset of transitions (n = 1,311), collected\nfrom multiple molecules across all conditions. Although individual traces – such as the example\nshown in Fig. 1e – may include events with closed-state lifetimes longer than 500 ms (or\nshorter), these fluctuations are fully incorporated into the overall statistical analysis.\nComparable heterogeneity in OppA closed-state dwell times has been reported previously;\nfor example, (de Boer et al, 2019) (Supplementary Fig. 2-1) observed variations of up to a factor of\nthree within a single smFRET trace at peptide concentration near the K .\nD\nIn response to the reviewer’s request, we now provide dwell-time histograms for all\nexperimental conditions use to derive the rates in Fig. 1f. These are included as Fig R1. The\nhistograms summarize open- and closed-state dwell times under each experimental condition.\nWe also note that temporal resolution is inherently limited by the acquisition speed of the\nmicroscope. Within this constraint, calculated closing rates show a clear linear dependence on\npeptide concentration. This relationship enables us to relate OppA closing kinetics to lumenal\npeptide concentrations, which are in excellent agreement with theoretical estimates.\nFig. R1: Dwell time histograms of OppA at different peptide concentrations. a, Open-state dwell-time\ndistributions showing progressively shorter open-state lifetimes with increasing concentrations of peptide\n(RRYQKSTEL). b, Closed-state dwell-time distributions demonstrating consistent closed-state lifetime across\nall peptide concentrations.\n8) The above problems persist in Figures 2-3 and pose a challenge to interpret data in figure 3.\nHow do the distributions of open/closed state of individual liposomes look like? If the main\nclaim to make is that 1 or 2 peptides get transported than it would be good to analyze the data\naccordingly and show that in some liposome the on/off-rate or FRET histogram support this\nwhile in other maybe not transport has occurred; currently the interpretation and possibilities\nare averaged out in both figures and no data/analysis of individual liposomes is shown. I highly\nrecommend to change this.\nReply: We fully agree with the reviewer that single-liposome-level analysis is essential for a\ncorrect interpretation of the data in Figure 2. By calibrating liposome size and correlating the\nfluorescence intensity of individual liposomes with FRET sensor responses, we can directly\nmonitor and quantify peptide transport events on a per-liposome basis.\ndistinguish liposomes that undergo one or two transport events from those that show no\ntransport. Accordingly, the variability in open/close-state distributions across individual\nliposomes is now captured, rather than averaged out, and we show representative cases\nillustrating the expected heterogeneity.\nA detailed description of the reconstitution strategy, imaging workflow, and per-liposome\nanalysis is provided in our response to point 12 (Reviewer #1) and in the revised Methods\nsection. These additions clarify the homogeneity of the preparation and demonstrate that the\nobserved transport events are robust at the single-liposome level.\n9) The liposome size distribution is heterogenous with a population of 200 nm diameter (40%),\nbut already this one has a substantial shoulder with 250 nm diameter (40%) and another large\none >300-350 nm (20%); thus, any concentration calculation from rates of blinking will be\ncompletely different for these liposome size (up to factors of 5-10!)\nReply: We fully agree with the reviewer that liposome size heterogeneity can strongly influence\n´concentration estimates. As detailed in our response to point 12 (Reviewer #1), we have now\nimplemented improved liposome preparation and size characterization, followed by refined on-\nsurface size calibration. These steps result in a substantially more homogeneous population of\nliposomes with diameters of ~100 nm diameter. This improved uniformity markedly reduces\nvariation in the lumenal volume and therefore minimizes differences in peptide concentration\nper liposome after transport.\n10) The estimation made in panel f to relate 4 μM to 1 and 6 μM to 2 peptides is incorrect and\nthus fundamental conclusions made in the paper are not supported/valid. The paper says\n“These lumenal peptide concentrations matched theoretical estimates for approximately one\nand two peptides in liposomes ranging of 200 ± 10 nm in diameter (Fig. 3g), …”. My own\nestimate of the calculations in excel below shows a fundamental problem of the idea of the\nauthors, i.e., to distinguish 1 or 2 peptide molecules but also use a heterogenous mixture of\nliposomes for this. In other words – currently it is not possible (explained below).\nReply: We acknowledge this limitation. To address it, we have added more detailed information\non liposome reconstitution, centrifugation, and size calibration. This workflow produces a\nuniform population of ~100 nm diameter liposomes. With this well-defined size, the theoretical\nlumenal concentration of a single peptide per liposome (~3.2 μM) aligns with the sensor’s K\nD\nvalue, allowing us to distinguish single- versus multiple-peptide transport events. These\nupdated methods and corresponding data are presented in the New Supplementary Fig S11\nand included in Fig. 3g.\n11) Arising problem 1: the liposome size should be smaller so that the sensor range with Kd of 1 μM\nis most sensitive to changes of 1 to 2 peptides, for this a diameter of 150 nm would be\nideal (see below)\nReply: Following this suggestion, we optimized our liposome preparation to achieve a mean\ndiameter of ~100 nm. At this size, the lumenal peptide concentration resulting from single-\npeptide occupancy falls within the optimal detection range of the FRET sensor (K ≈ 1 µM),\nD\nthereby ensuring high sensitivity to single-peptide events while maintaining robust and reliable\nsignal detection. This adjustment also minimizes variability due to liposome size heterogeneity.\n5\n12) Problem 2: the liposome size distribution together with the averaging the authors do will not\nallow them to make any conclusion since the differences between liposomes of 200 nm and\n350 nm has tremendous differences in the molecule number per vesicle (10 for 4 μM for 200\nnm and 54 for 350 nm); in principle the best solution would be to calibrate the liposome size\nin each image using an intensity-based size measure (e.g., with a lipid dye) relating the intensity\ndistribution to the size distribution.\nReply: We fully agree with this point and have implemented the following improvements:\n(i) Liposome size determination on the microscope surface: Liposomes were stained with\nthe lipophilic dye DiD and immobilized on PEG-biotin surfaces via nanobody\ninteractions to capture single TmrAB transporters in an uptake-competent orientation.\nFluorescence intensity of individual liposomes was used to infer their diameters,\nconfirming homogeneity and revealed that the majority of captured liposomes at the\nsurface have a diameter of ~100 nm (New Fig. 3g and New Supplementary Fig. 11).\n(ii) Implications for peptide quantification: With this uniform liposome population, single-\npeptide concentrations correspond to ~3.2 μM, enabling accurate analysis of single-\ntransport events and eliminating artifacts from averaging across heterogeneous\nliposome sizes. These improvements ensure that the single-liposome FRET data reliably\nsupports our conclusions regarding single-peptide transport events.\n13) The author also do not provide another absolutely essential control, which is related to their\napproach. Since they have a label-free approach (from the viewpoint of the transporter) they\nhave no readout to see how many transporters per liposome (with varying size) actually caused\nthe change of sensor read-out. What is needed: label the transporter and reconstitute it at the\nsame protein-to-lipid ratio as in the normal assays and count how many transporters are in\nthe liposome to understand this effect.\nReply: We appreciate the reviewer’s suggestion. To address this, we carefully controlled the\nprotein-to-lipid ratio during reconstitution such that, statistically, only one transporter is\npresent per ~10 liposomes. Consequently, the majority of liposomes are empty and removed\nduring washing, leaving a small population containing a reconstituted TmrAB.\nAdditionally, to directly assess transporter copy number per liposome, we labeled TmrABWT\ncontaining a single cysteine with AF647. Using the same reconstitution protocol, one-step and\ntwo-step photobleaching events were counted (New Supplementary Fig. 6). This analysis\ndemonstrates that 95.6% of liposomes contain a single reconstituted transporter.\nThis stochastic reconstitution approach, combined with orientation-selective immobilization,\nensures that the observed FRET responses predominantly arise from single transporters per\nliposome.\n14) Another possibility would be to perform control experiments in which 1 or 2 peptides are\nreconstituted into the liposomes (without transporter) but with sensor to extract kinetics\noff/on.\nReply: We thank the reviewer for this suggestion. However, due to the stochastic nature of\nencapsulation, it is not feasible to prepare liposome populations containing exactly one or two\npeptides without introducing substantial heterogeneity. In contrast, our TmrAEQB\nsingle-turnover assay inherently restricts peptide transport to single events per liposome within\nthe assay window, providing a well-defined system to accurately quantify single-peptide\ntranslocation.\n6\n15) To say it clearly: currently the paper cannot support any claims and interpretations made\nfurther on the mechanistic side since the controls are lacking, and importantly: the calculations\nand assumptions on the ability of the assay to see single-peptide transport are not correct (and\nnot provided in the manuscript, so it is difficult to see where the error comes from or if\ndifferent assumptions were made).\nReply: We thank the reviewer for this important comment. To address these concerns, we have\nincluded extensive additional controls, expanding the Supplementary Information from Fig. 10\nto Fig. 17. Mechanistic interpretations based on single-molecule results are further\ncorroborated by orthogonal approaches, including cryo-EM, radioactivity-based ATPase\nmeasurements, and ensemble transport assays based on LC-MS.\nRegarding the theoretical calculations of single-peptide encapsulation, the original calculations\nwere correct. To enhance clarity and transparency, we have now explicitly provided these\ncalculations in the Methods section. With the updated, well-defined size of surface-immobilized\nliposomes (~100 nm diameter), the theoretical lumenal concentration of a single peptide aligns\nclosely with the observed smFRET data. These improvements confirm that our experimental\nsetup reliably reports single-peptide transport events, thereby supporting the mechanistic\nconclusions presented in the manuscript.\nReviewer #2 (Remarks to the Author)\nThe manuscript by Nocker is probing the role of Mg2+ in either coordinating ATP or facilitating\nhydrolysis and subsequent substrate transport of the ABC transporter TmrAB. The authors use a\nsmFRET approach of TmrAB reconstituted in liposomes to monitor peptide transport by utilizing\nthe OppA as reporter. They evaluated their system in the presence of ATP-Mg2+ and peptide, with wt or\nATPase deficient TmrAB EQ mutant. They also pretreated their proteoliposomes with EDTA and added\nATP-EDTA, and they observed that a single peptide transport event takes place. Based on these\nobservations they conclude that ATP alone is capable to drive transport but not resetting the\ntransporter for a subsequent transport event. Finally, using cryo-EM analysis of TmrAB with ATP-EDTA,\nthey show that the transporter is capable of adopting the outward facing conformation without Mg2+\nfurther validating their findings.\nThe work is built on the authors expertise to study TmrAB. The experiments have been executed to\nhigh standards and with fairly sound conclusions. Some of the results such as the transport of single\npeptide is just a reconfirmation of their previous work about TmrAB transporting a single peptide per\ntransport event. Debugging the role of Mg2+ is the novelty of this study.\nReply: We sincerely thank Reviewer #2 for the encouraging and thoughtful evaluation of our work. We\nare pleased that the reviewer recognizes the rigor and high quality of our experiments and finds our\nconclusions sound. We particularly appreciate the acknowledgement of our expertise with TmrAB and\nare pleased that the novelty of our study—clarifying the role of Mg²⁺ in ATP-driven transport—is\nrecognized as an important advancement to understanding the mechanistic basis of single-peptide\ntransport.\nSpecific Comments:\n1) My main concern is the lack of discussion on the presence of OppA species that do not show\nhigh FRET upon a transport event in all their experiments; under all conditions with ATP and\npeptide, the plots show a bimodal behavior that the authors have not addressed and it\npresents a limitation of using OppA as the reporter rather than labelling TmrAB directly. This\nis the biggest weakness of this manuscript. The plots show a significant portion of OppA\nmolecules not binding the peptide even under conditions of saturation. This could either be\ndue to the presence of more than one TmrAB in the liposome (there is no evidence for this) or\n7\nencapsulation of more than one OppA or a more complex transport kinetic model that needs\nfurther investigation.\nReply: We thank the reviewer for raising this important point. The observed histograms reflect\nboth low- and high-FRET populations of OppA. Peptides are released after ~0.5 s, resulting in a\nshift from high to low FRET, and the peptide concentration determines the rate of reassociation.\nA small low FRET population is therefore always present, and achieving complete sensor\nsaturation requires multiple peptide translocation events at very high peptide concentrations.\nIn our experiments, typically only one or two translocation events occur per liposomes –\ninsufficient to fully shift all OppA sensors into the high-FRET state. To demonstrate this, we\nperformed an additional experiment with three sequential translocation events through\nTmrAEQB (Fig. R2), which shows near-complete saturation of the sensor and a corresponding\nshift to the high FRET state.\nA small fraction of OppA molecules remain static in the low-FRET state, likely due to damaged\nsensors or transporters. Importantly, our Hidden Markov Model (HMM) analysis excludes these\nstatic traces, considering only dynamic traces that exhibit transitions upon peptide binding. This\nensures that the kinetic rates reported are derived solely from OppA molecules actively\nreporting transport events. By contrast, the histograms include both static and dynamic traces,\nwhich explains the persistent low-FRET populations.\nWhile direct labeling of TmrAB would provide complementary information, it would not allow\ndirect observation of substrate transport, which is the key advantage of using a single-molecule\nreporter. As further validation, liposome experiments using α-hemolysin pores (New\nSupplementary Fig. 6e) show that 90-95% of OppA molecules are capable of binding peptide\nand shifting to the high-FRET state, confirming the sensor’s functionality.\nIn addition, we provide now evidence that single TmrAB transporters are reconstituted in the\nliposomes by labeling TmrAB with a single AF647 fluorophore and counting one-step and two-\nstep photobleaching events (New Supplementary Fig. 6a-c), as noted in our response to\nReviewer #1 (comment 13). This demonstrates that the majority of liposomes contain only one\nfunctional transporter, ruling out multiple transporter events.\nFig. R2: OppA exhibit a near-complete shift towards the high-FRET state after three sequential peptide\ntranslocation steps by TmrAEQB (n = 192).\n2) The legend for Fig. 3f on ‘transition kinetics’ is not relevant to TmrAB as the FRET is only\nreporting on OppA binding kinetics which is not the focus of this study. This should be changed.\nReply: We thank the reviewer for this comment. The figure legend of Fig. 3f has been updated\nto read: “Distinct OppA peptide-binding kinetics”. This change clarifies that the FRET signal\n8\nreports on OppA sensor binding rather than TmrAB conformational changes, ensuring accurate\ninterpretation of the data.\n3) Fig. 2 would also benefit from having an HMM analysis table as Fig. 3, and make comparisons\nbetween the wt and mutant protein in the discussion.\nReply: We thank the reviewer for this suggestion. While HMM analysis is highly informative for\nTmrAEQB, a direct comparison with TmrABWT is not feasible due to differences in experimental\nconditions:\n(i) Temperature difference: TmrABWT transport was initiated on the microscope at 30 °C,\nwhereas TmrAEQB assays were conducted at 45 °C, which substantially affects transport\nactivity (see also Reviewer #3, point 7).\n(ii) Measurement setup differences: TmrABWT transport was recorded over time in 10-min\nintervals, limiting the number of observed molecules. In contrast, TmrAEQB\nmeasurements were performed as 5 min end-point reactions, followed by 1 h imaging,\nenabling sufficient molecule counts for robust HMM analysis.\nGiven these difference, Fig. 2 primarily demonstrates sensor’s capability for TmrABWT, while\nHMM analysis is applicable and statistically meaningful only for TmrAEQB.\n4) Under the cryo-EM results section, the manuscript would benefit by providing a comparison\nof the NBDs with and without Mg2+; ie do the side chains adopt different rotamers? They\nshould also provide rmsd values for the two structures. Are there any conformational changes\nwithin the NBD/coupling helix region that the absence of Mg2+ can induce?\nReply: We thank the reviewer for this insightful suggestion. We have now directly compared\nthe NBDs of TmrAEQB bound to ATP in the absence of Mg2+ with the previously reported Mg-ATP\ncomplex (PDB 6RAI; EMD-4776), as shown in New Supplementary Fig. 14. The RMSD between\nthe two structures is 0.51 Å across all Cα atoms, indicating that Mg2+ removal has negligible\nimpact on the overall global structure of the ATP-bound outward-facing occluded (OFoccluded)\nstate of TmrAEQB.\nNonetheless, subtle rearrangements are observed within the nucleotide-binding sites: residues\ncoordinating Mg2+ (TmrAT400, TmrBQ419) adopt slightly different rotamers in its absence, as\nillustrated in New Supplementary Fig. 14b–e. No significant conformational changes are\ndetected in the NBD-coupling helix regions, suggesting that Mg2+ removal does not\nsubstantially perturb the structural elements mediating NBD-TMD communication.\n5) The statement in page 10 ‘Yet our work uncovers a previously overlooked Mg2+- independent\nATP-bound state that supports single-turnover transport.’ is confused with their previous\nsentence and there is not much relevancy for transporters which are greatly uncoupled for\nATP-dependent substrate transport.\nReply: We thank the reviewer for this helpful comment. To avoid confusion and ensure the\nmanuscript remains focused on the relevant mechanistic aspects to TmrAB transport, we have\nremoved this sentence from the revised manuscript.\n6) Supplementary Table 1 is reporting the EQ mutant structure but there is no figure of its ATP\nbinding site as for the wt and any discussion beyond the difference maps. Supplementary Fig\n8 should show the process of both wt and EQ mutant separately.\nTmrAEQB and its nucleotide-binding sites are now included as New Supplementary Fig. 14.\nAdditionally, the cryo-EM workflow for TmrAEQB has been added as New Supplementary\nFig. 13, allowing a direct comparison with the TmrABWT dataset. These additions provide a\nclearer visualization of structural similarities and differences between the WT and EQ mutant.\nReviewer #3 (Remarks to the Author)\nSingle-molecule dynamics reveal ATP binding alone powers substrate translocation by an ABC\ntransporter.\nThe authors show that binding of ATP is sufficient for a half turnover of the peptide ABC transporter\nTmrAB, i.e. the transport of a peptide from cis to trans. Mg-ATP is required for hydrolysis and the other\nhalf turnover, which resets the system for the next translocation event. Thus, the conformational\nswitch from inward-facing to outward-facing by ATP (without Mg2+ ions) is a key finding of this paper,\nwhich has been postulated before but may not have demonstrated as convincingly as done for TmrAB\nin this paper. Perhaps, the authors should summarize in their discussion to what extent the ATP-driven\nhalf turnover is unique for TmrAB (or heterodimeric ABCs), and indicate other studies where this has\nbeen shown directly or indirectly or ruled out. Now only computational studies and CFTR (special case)\nare mentioned in the Discussion section.\nOverall, this is a beautifully executed single-molecule study on an important bacterial homologue of\nthe human TAP system. The work is important and well presented. We have a few questions and minor\ncomments for the authors to consider:\nReply: We sincerely thank Reviewer #3 for the thoughtful and encouraging evaluation of our work. We\nare delighted that the reviewer considers our single-molecule study to be “beautifully executed” and\nrecognizes the importance of TmrAB as a model for the human TAP system. We greatly appreciate\nthe acknowledgment that our findings convincingly demonstrate ATP-driven half-turnover and the\nassociated conformational switch in TmrAB, underscoring the novelty and mechanistic relevance of\nour study.\nIn response to the reviewer’s helpful suggestion, we have expanded the Discussion section to include a\nbroader comparison of ATP-driven half-turnover mechanisms across ABC transporters. Specifically, we\nnow summarize previous biochemical and structural studies that report related phenomena in\nheterodimeric ABC exporters and highlight where such transitions have been directly or indirectly\nobserved, or ruled out. These additions place our findings in a wider mechanistic context and clarify the\nextent to which the ATP-driven half-turnover observed in TmrAB may represent a general feature of\nasymmetric ABC transporters.\nSpecific Comments:\n1) Figure 1d reports K = 0.9 µM (text indicates values from 0.2-0.9 µM) for RRYQKSTEL binding\nD\nto OppA; Figure 1f reports k and τ, from which the K can be calculated, which then yields\non D\nK = 7 µM. What is the explanation for the approximately 10-fold difference in binding\nD\nconstant?\nReply: We thank the reviewer for raising this important point. The apparent difference between\nthe K values arises from differences in experimental conditions and subsets of data analyzed.\nD\nEnsemble measurements (tryptophan quenching, K = 0.22 ± 0.04 µM; ensemble FRET,\nD\nK = 0.3 ± 0.1 µM) report the thermodynamic equilibrium K . of OppA for peptide RRYQKSTEL in\nD D\nsolution. Single-molecule experiments with surface-immobilized OppA yield a slightly higher K\nD\n(0.9 ± 0.2 µM), likely reflecting minor effects of immobilization on peptide accessibility or\nprotein conformational flexibility.\nD on off\nrates derived only from dynamic traces showing FRET transitions. Static molecules that remain\nin the high-FRET state are excluded, which can result in an overestimation of K . Additionally,\nD\npeptide rebinding events may influence the observed kinetic rates.\nSuch differences between thermodynamic and kinetic K values are well-documented; for\nD\nexample, de Boer and colleagues observed that OppA binding to peptide RPPGFSPFR showed a\nthermodynamic K of 5 ± 3 µM (ITC) versus a kinetic K of 14 ± 5 µM (smFRET) (Boer et al.,\nD D\n2019).\nOverall, the ~10-fold variation is consistent with expectations and reflects the intrinsic\ndifferences between ensemble equilibrium and single-molecules kinetic analyses.\n2) The internal volume of the vesicles is calculated for a diameter of 160 nm, whereas the peak\nin the vesicle concentration is around 200 (Fig. 3g). Why is 160 nm chosen, and how does the\ndistribution of vesicles sizes (from 100 to 500 nm) affect the analysis of the data (e.g. in the\ncontext of max 1 OppA and 1 TmrAB per vesicle, and both proteins need to be in the same\nvesicle)?\nReply: We thank the reviewer for this insightful question. In the section “Rationale of single-\nsubstrate sensors in liposomes”, a vesicle diameter of 160 nm was used as a representative\nexample to illustrate the underlying calculation and concept. At this size, the internal volume\ncorresponds to approximately 2 aL, resulting in an effective peptide concentration of about\n1 µM for a single peptide molecule. This choice was made for conceptual clarity.\nWe agree that the liposome diameter strongly influences lumenal peptide concentration. For\nexample, vesicles of 100 nm, 200 nm, and 300 nm in diameter correspond to single-peptide\nconcentrations of ~3.2 µM, 0.4 µM, and 0.1 µM, respectively. Larger vesicles (>300 nM) are\nmore likely to contain multiple OppA molecules and are therefore excluded from analysis.\nAs noted in our response to Reviewer #1 (comment 12), although the vesicle population ranges\nfrom 100 to 300 nm, the majority of vesicles immobilized at the surface are ~100 nm in\ndiameter as confirmed by New Supplementary Fig. 11. At this size, the lumenal peptide\nconcentration closely matches the theoretical estimate for one peptide per vesicle, assuming a\nsingle-substrate translocation event.\nIn addition, to directly assess transporter copy number per liposome, we labeled TmrABWT\ncontaining a single cysteine with AF647. Using the same reconstitution protocol, one-step and\ntwo-step photobleaching events were counted (New Supplementary Fig. 6). This analysis\ndemonstrates that 95.6% of liposomes contain a single reconstituted transporter (see comment\n13 to Reviewer #1).\nConsequently, the majority of analyzed liposomes contain one OppA sensor and one TmrAB\ntransporter, enabling accurate single-peptide transport measurements.\n3) Fig. S4 shows that nanobody binding does not affect the activity of TmrAB. Since a high protein-\nto-lipid ratio is used, what is the evidence that the reported activity is actually transport and\nnot peptide binding? The same applies to the data of SFig.6. It should be possible to\ndiscriminate binding from transport by comparing the number of peptides translocated with\nthe number of TmrAB molecules in the membrane.\nReply: We thank the reviewer for raising this important point. In both the FACS transport assay\n(Supplementary Fig. 7) and the LC-MS transport assay (Supplementary Fig. 9), a clear ATP-\ndependent peptide accumulation is observed relative to the ADP control, confirming that the\nsignal reflects active transport rather than surface binding. In the LC-MS assay, residual peptide\n11\nbinding artifacts are further removed by a carbonate wash step (100 mM Na CO , pH 11.5),\n2 3\nwhich efficiently removes peptides adhering to the liposome surface.\nIn the smFRET-based assay, transport is directly monitored via the lumenal OppA sensor, which\nshows a distinct shift from low-FRET to high-FRET upon peptide translocation. If peptides were\nmerely binding TmrAB without being transported, no FRET change would occur, as verified in\nthe ADP control experiments (Fig. 2b and 3b). Additionally, a control experiment where peptide\nis added after the first translocation event in the absence of Mg-ATP (Supplementary Fig. 10f)\nshows no further FRET change, demonstrating that peptide movement into the lumen is strictly\nATP-dependent.\nNotably, the protein-to-lipid ratio in smFRET experiments was very low (1:10,000 w/w),\nmeaning that, statistically, only one in ten liposomes contains a single TmrAB complex,\nminimizing the likelihood of multiple transporters per vesicle. By contrast, ensemble assays\nused higher ratios (1:20 w/w) to increase the overall transport signal.\nTaken together, the combination of smFRET, LC-MS, and FACS assays provides robust evidence\nthat the signals reflect true peptide transport rather than mere binding events.\n4) Do non-hydrolysable ATP analogues also trigger the half turnover of TmrAB?\nReply: We thank the reviewer for this insightful question. To address it, we performed\nexperiments using the non (very slow)-hydrolysable ATP analog AMP-PNP, and the results are\nnow included as New Supplementary Fig. 17. These experiments demonstrate that AMP-PNP\ndoes not trigger half-turnover or peptide transport by TmrAB, indicating that ATP binding alone,\nrather than analogues, is required to drive a single translocation event.\n5) How was OppA encapsulated in the vesicle lumen? Page 23 only describes the membrane\nreconstitution of TmrAB.\nReply: We thank the reviewer for the comment. Although the manuscript primarily describes\nTmrAB membrane reconstitution, OppA was co-added to the detergent-destabilized liposomes\nduring this process. The transient openings created in the destabilized liposomes allow OppA\nto be encapsulated in the vesicle lumen concurrently with TmrAB incorporation. This co-\nreconstitution strategy ensures that the lumenal OppA sensor is present and functional for\nmonitoring peptide transport.\n6) Fig. 2a and 2b show a high FRET peak, which is not seen in Fig. 3a and 3b. Is this endogenously\nbound peptide in this particular preparation of OppA?\nReply: We thank the reviewer for this observation. The high-FRET peak in Fig. 2a and 2b arises\nfrom endogenously bound ligands in the OppA preparation. Even in the absence of added\npeptide and Mg-ATP, a small fraction of OppA remains in a static high-FRET state. The extent of\nthis proportion strongly depends on the protein purification protocol, particularly on the\nefficiency of removing endogenous ligands via partial unfolding and subsequent refolding with\nguanidine hydrochloride. Optimized purification, as employed for the data in Fig. 3, minimizes\nthis high-FRET fraction, resulting in a predominantly low-FRET apo state suitable for transport\nassays.\n7) Figure 2c shows the time dependence of peptide translocation (0-10 and 11-20 min) for\nwildtype TmrAB, but this is not shown for the EQ mutant in Figure 3c or 3d. Shouldn’t it be an\norder of magnitude slower?\nTmrAEQB exhibits ~1000-fold lower transport activity than wild-type TmrAB. However, the\ndataset in Fig. 2 (TmrABWT) and Fig. 3 (TmrAEQB) are not directly comparable due to differences\nin experimental conditions.\nThe wild-type TmrAB experiments (Fig. 2) were performed at 30 °C, where transport proceeds\nslowly enough for time-resolved smFRET imaging. At higher temperatures, wild-type transport\nbecomes too rapid to resolve individual events. In contrast, the TmrAEQB experiments (Fig. 3)\nwere performed at 45 °C: pre-heated imaging buffer was applied to immobilized liposomes for\n5 min, followed by washing to stop activity before imaging. This setup allows accumulation of\nsufficient single-turnover events accumulated for quantitative analysis despite the mutant’s\nslow kinetics.\nTo directly address the reviewer’s point, control experiments with TmrAEQB at 30 °C\n(Supplementary Fig. 10d, e) show a larger low-FRET population and a reduced high-FRET\npopulation compared with the 45 °C dataset, confirming that transport by TmrAEQB is\nsubstantially slower at lower temperatures.\n8) Figure 6 suggests that 1 ATP is hydrolyzed per peptide translocated. Is there evidence for this\ncontention; they also write on page 10 that wildtype TmrAB exhibits futile hydrolysis of ATP.\nReply: We thank the reviewer for raising this important point. The ATP-to-peptide coupling\nratio indeed depends on the transporter variant and the biochemical conditions.\nFor TmrAEQB, our data support an approximately 1:1 coupling ratio: one ATP hydrolyzed per\npeptide translocated. As shown in Fig. 4f, TmrAEQB hydrolyzes ~0.25 ATP/min in the presence\nof MgCl , corresponding to ~1.2 ATP molecules during the 5-min transport window, which is\n2\nconsistent with the single-peptide translocation events observed by smFRET. This 1:1 ratio was\nalso previously observed (Stefan et al, 2020).\nFor wild-type TmrAB, under MgCl conditions, ATP hydrolysis occurs at ~1 ATP/s (Fig. 4e),\n2\nreflecting substantial futile turnover, as is common for many ABC exporters (Bock et al, 2019).\nUnder these conditions, the ATP/peptide coupling ratio is therefore not 1:1.\nUnder Mg2+-free (EDTA) conditions, ATP hydrolysis is reduced to background levels (Fig. 4e),\nyet single-peptide transport still occurs (Supplementary Fig. 16c, e). In this scenario, the\neffective coupling ratio approaches one ATP binding event per peptide translocated, consistent\nwith the half-turnover mechanism described in the manuscript.\nIn summary, a near 1:1 ATP-to-peptide ratio is supported for the TmrAEQB system and for\nTmrABWT under Mg2+-free conditions, whereas wild-type TmrAB under normal Mg2+ conditions\nexhibits substantial uncoupled ATP hydrolysis.\n9) Page 28: It is not entirely clear how FRET traces were selected for the FRET distributions. Were\ntraces without dynamics discarded? What were the criteria?\nReply: We thank the reviewer for this question. All smFRET traces were initially processed using\nDeepFRET, which assigns a per-trace confidence score based on a pretrained neural network\nclassifier. Following the approach of Thomsen et al., we applied a confidence threshold of 80%,\nand only traces exceeding this value were retained for further analysis (Thomsen et al, 2020).\nFor the FRET histograms, we included both static and dynamics traces that passed this\nconfidence threshold, ensuring that the histograms represent the full molecular population. For\nthe HMM analysis, only dynamic traces were used, as these contain state transitions necessary\nfor extracting dwell times and kinetic parameters. Static traces lack transitions and thus do not\ncontribute meaningful kinetic information.\nThis two-step approach ensures that the FRET distributions capture the overall population\nwhile the kinetic analysis relies exclusively on mechanistically informative traces.\n13\n10) Fig. S3a and b: Why a double peak in the SEC profile for wildtype TmrAB, which is not seen for\nthe EQ protein.\nReply: We thank the reviewer for this observation. SDS-PAGE analysis confirmed that both\npeaks in the SEC profile of TmrABWT correspond to properly assembled transport complexes.\nThe first peak (F1) represents DDM micelles containing TmrAB dimers, whereas the second\npeak (F2) corresponds to micelles containing a single TmrAB complex. The double peak reflects\na time-dependent equilibrium between singly and doubly occupied micelles.\nTo clarify, we repeated the SEC purification and updated the figure; the front peak (F1) is now\nsignificantly reduced. A statement explaining this behavior has been added to the legend of\nSupplementary Fig. 5.\n11) Fig. S7a: The high FRET state after one translocation event is similar to the high FRET state after\ntwo translocation events of Fig. 3d. Similarly, in Fig. S10e, the high/low FRET ratio is lower after\ntranslocation event 2 than after 1 (and even after 3 events it may still be lower than after 1).\nA brief description on variations between experiments and reproducibility or other\nexplanation would be helpful.\nReply: We thank the reviewer for this observation. The slightly different high-FRET populations\nobserved in previous Supplementary Fig. S7a, now Supplementary Fig. 10a and\nSupplementary Fig. 16c primarily reflect experimental variability and statistical sampling\ndifferences.\nFirst, Supplementary Fig. 10a was recorded in the presence of the ionophore valinomycin,\nwhich is necessary to dissipate the membrane potential, may subtly influence liposome\nintegrity or local membrane properties, modestly affecting the OppA smFRET sensor (Gutiérrez-\nPineda et al, 2021; Hussein & Sarles, 2025; Rose & Jenkins, 2006).\nSecond, sample size differences contribute to apparent variations. Fig. 3 datasets included\n~800 molecules per condition, yielding smooth, robust histograms, whereas the supplemental\nexperiments contained fewer molecules (Supplementary Fig. 10a: n = 148; Supplementary\nFig. 16c, translocation event 1: n = 158; translocation event 2: n = 90, translocation event 3:\nn = 246), naturally resulting in fluctuations in the high-/low-FRET populations.\nImportantly, despite these differences, all datasets consistently support the same mechanistic\nconclusion: under ATP-EDTA conditions, TmrAB does not perform additional peptide\ntranslocation events after the initial ATP-driven half-turnover. The observed variations reflect\nnormal sampling variability rather than inconsistencies in transporter behavior.\n12) The description of the legend of Fig. S10g (Hidden Markov Model) is ambiguous and needs\nsome rewriting.\nReply: We thank the reviewer for this suggestion. The legend has been revised and the figure\nrenumbered as Supplementary Fig. 16e. It now clearly states that Hidden Markov Model\n(HMM) analysis was applied exclusively to dynamic OppA traces exhibiting FRET transitions,\nwhile static traces without observable transitions were excluded from this analysis to extract\ndwell time accurately.\nBlanco M, Walter NG (2010) Analysis of complex single-molecule FRET time trajectories. Methods\nEnzymol 472: 153–178. doi:10.1016/S0076-6879(10)72011-5\nBock C, Zollmann T, Lindt KA, Tampé R, Abele R (2019) Peptide translocation by the lysosomal ABC\ntransporter TAPL is regulated by coupling efficiency and activation energy. Sci Rep 9: 11884.\ndoi:10.1038/s41598-019-48343-6\nCiftci D, Huysmans GHM, Wang X, He C, Terry D, Zhou Z, Fitzgerald G, Blanchard SC, Boudker O (2020)\nSingle-molecule transport kinetics of a glutamate transporter homolog shows static disorder. Sci\nAdv 6: eaaz1949. doi:10.1126/sciadv.aaz1949\nde Boer M, Gouridis G, Vietrov R, Begg SL, Schuurman-Wolters GK, Husada F, Eleftheriadis N, Poolman\nB, McDevitt CA, Cordes T (2019) Conformational and dynamic plasticity in substrate-binding\nproteins underlies selective transport in ABC importers. eLife 8: 44652. doi:10.7554/eLife.44652\nFitzgerald GA, Terry DS, Warren AL, Quick M, Javitch JA, Blanchard SC (2019) Quantifying secondary\ntransport at single-molecule resolution. Nature 575: 528–534. doi:10.1038/s41586-019-1747-5\nGutiérrez-Pineda E, Andreozzi P, Diamanti E, Anguiano R, Ziolo RF, Moya SE, Rodríguez-Presa MJ, C. C\n(2021) Effects of valinomycin doping on the electrical and structural properties of planar lipid\nbilayers supported on polyelectrolyte multilayers. Bioelectrochemistry 138: 107688.\nHolden SJ, Uphoff S, Hohlbein J, Yadin D, Le Reste L, Britton OJ, Kapanidis AN (2010) Defining the\nlimits of single-molecule FRET resolution in TIRF microscopy. Biophys J 99: 3102–3111.\ndoi:10.1016/j.bpj.2010.09.005\nHusada F, Bountra K, Tassis K, de Boer M, Romano M, Rebuffat S, Beis K, Cordes T (2018)\nConformational dynamics of the ABC transporter McjD seen by single-molecule FRET. EMBO J 37\ndoi:10.15252/embj.2018100056\nHusada F, Gouridis G, Vietrov R, Schuurman-Wolters GK, Ploetz E, de Boer M, Poolman B, Cordes T\n(2015) Watching conformational dynamics of ABC transporters with single-molecule tools.\nBiochem Soc Trans 43: 1041–1047. doi:10.1042/BST20150140\nHussein EA, Sarles SA (2025) Spontaneous depletion of valinomycin-mediated ion current in droplet\ninterface bilayers. Langmuir 41: 26697–26704. doi:10.1021/acs.langmuir.5c03024\nKosmidis E, Shuttle CG, Preobraschenski J, Ganzella M, Johnson PJ, Veshaguri S, Holmkvist J, Moller\nMP, Marantos O, Marcoline F, Grabe M, Pedersen JL, Jahn R, Stamou D (2022) Regulation of the\nmammalian-brain V-ATPase through ultraslow mode-switching. Nature 611: 827–834.\ndoi:10.1038/s41586-022-05472-9\nRose L, Jenkins ATA (2006) The effect of the ionophore valinomycin on biomimetic solid supported\nlipid DPPTE/EPC membranes. Bioelectrochemistry 70: 387–393.\nStefan E, Hofmann S, Tampé R (2020) A single power stroke by ATP binding drives substrate\ntranslocation in a heterodimeric ABC transporter. eLife 9: 55943. doi:10.7554/eLife.55943\nThomsen J, Sletfjerding MB, Jensen SB, Stella S, Paul B, Malle MG, Montoya G, Petersen TC, Hatzakis\nNS (2020) DeepFRET, a software for rapid and automated single-molecule FRET data classification\nusing deep learning. eLife 9: 60404. doi:10.7554/eLife.60404\nWang L, Johnson ZL, Wasserman MR, Levring J, Chen J, Liu S (2020) Characterization of the kinetic\ncycle of an ABC transporter by single-molecule and cryo-EM analyses. eLife 9\ndoi:10.7554/eLife.56451\nReply to Reviewer Response: I have checked both the comments of Reviewer 1 and the responses of the\nauthors. Reviewer #1 has valid points. The authors answered the question partially satisfactorily, e.g. see\npoints 9 and 12. The largest concern raised is in the heterogeneity of the vesicles and the limitations of\ncurrent reconstitution methods to obtain unilamellar vesicles. In my opinion, the authors couldn’t have\ndone much better than they did. Reviewer 1 has a valid point that casts doubt on some of the\nconclusions in the article. I believe the main conclusion, which is reflected in the article's title, remains\nvalid. Please see below for specific comments.\nWe sincerely thank the reviewer for the time, care, and thoughtful attention devoted to evaluating both\nReviewer #1’s comments and our responses. We truly appreciate the acknowledgement of the efforts we\nmade to address the points raised, particularly regarding vesicle heterogeneity and reconstitution\nlimitations. In response, we have added a short paragraph in the Discussion to explicitly acknowledge this\nlimitation while clarifying how it is mitigated:\n“…Finally, while liposome size control after reconstitution is inherently limited, the applied protocols\nreliably generate predominantly unilamellar vesicles (DOI:10.1038/nprot.2007.519). Importantly, the\nassay design–using very low transporter-to-lipid ratios and orientation-specific surface immobilization–\nselectively removes empty, oversized, and non-functional liposomes, resulting in a highly enriched\npopulation of function proteoliposomes with an apparent size of ~100 nm (Supplementary Fig. 11).\nWe hope this addition addresses the concern and further strengthens the clarity and rigor of our\nmanuscript.\nReply to Reviewer Response Point 9 and 12:\nLiposomes were prepared by thin-film hydration, followed by sonication, five freeze-thaw cycles, and\nextrusion through a 100 nm polycarbonate membrane prior to reconstitution. Freeze-thaw cycling is\nknown to substantially reduce multi-lamellarity, yielding approximately 91% unilamellar vesicles, while\nextrusion further disrupts the remaining multilamellar structures, as demonstrated previously (DOI:\n10.1021/acs.langmuir.8b04256;DOI: 10.1021/acs.langmuir.8b04256; DOI: 10.1016/s0378-5173(01)00721-\n9; DOI:10.1016/0378-5173(87)90139-6). In addition, protein reconstitution was performed following the\nwell-established protocol by Geertsma et al. (2008), which is reported to produce unilamellar\nproteoliposomes with a low fraction of multilamellar vesicles (DOI: 10.1038/nprot.2007.519.).\nWe acknowledge that the multi-lamellarity is not directly probed by the fluorescence readout itself.\nHowever, the assay design strongly disfavors contributions from multilamellar vesicles. Following\nextrusion through pores <200 nm, only ~9% of bilamellar vesicles are expected to remain. Moreover, the\nexperimental configuration requires that OppA be encapsulated within the same vesicle as TmrAB for\ntransport activity to be detected. Multilamellar vesicles, incorrectly oriented transporters, or liposomes\nlacking functional encapsulation of OppA are therefore intrinsically excluded from the functional and\nsingle-molecule analyses. Additionally, non-tethered liposomes were removed by washing, further\nenriching for assay-competent vesicles.\nRegarding liposome size, nanoparticle tracking analysis (NTA) was performed on samples directly after\nreconstitution, a method known to overestimate the contribution of larger particles and to broaden the\napparent size distribution. In contrast, samples used for single-molecule FRET measurements underwent\nan additional centrifugation step (2,000 × g, 2 min) prior to surface immobilization. This step efficiently\nremoves large and aggregated liposomes, explaining the narrower size distribution observed under\nsingle-molecule conditions. Furthermore, highly fluorescent aggregates (e.g., the bright spot visible in\nSupplementary Fig. S11) were excluded from analysis based on DiD membrane staining. As a result, the\nliposome population analyzed at the surface is effectively enriched for ~100-nm vesicles.\nTaken together, the photobleaching statistics, established liposome preparation protocols, and the\nintrinsic selectivity of the assay design collectively support the conclusion that the analyzed\nproteoliposomes predominantly contain a single transporter embedded in unilamellar vesicles.\nReviewer Response to Point 13: The authors have done the requested experiment. I believe that the\nmajority of the vesicles with TmrAB have 1 transport protein per liposome, however it is hard to believe\nthat the accuracy is 96.5%.\nThe reported value of 95.6% was calculated based on 2,088 individual photobleaching traces. Of these,\n1,997 traces (95.6%) displayed a single-step photobleaching event, consistent with the presence of a\nsingle transporter per liposome, while only 91 traces (4.4%) showed a two-step photobleaching event,\nindicative of two transporters per liposome (Supplementary Fig.6). These results are in good agreement\nwith the chosen reconstitution conditions, which were designed to yield, on average, one transporter per\nten liposomes, resulting in a population dominated by empty liposomes and a low probability of multiple\ntransporters per vesicle. We therefore conclude that the vast majority (>90%) of transporter-containing\nliposomes harbor only a single reconstituted transporter."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have addressed all my queries and the clarity of the revised manuscript has improved.", "reviewer_3": "I am impressed by the thoroughness of the rebuttal and the way the authors have addressed the comments and suggestion sof the three reviewers. The rebuttal is a valuable addition to the manuscript. I have no further comments.", "reviewer_4": "I have checked both the comments of Reviewer 1 and the responses of the authors. Reviewer #1 has valid points. The authors answered the question partially satisfactorily, e.g. see points 9 and 12. The largest concern raised is in the heterogeneity of the vesicles and the limitations of current reconstitution methods to obtain unilamellar vesicles. In my opinion, the authors couldn’t have done much better than they did. Reviewer 1 has a valid point that casts doubt on some of the conclusions in the article. I believe the main conclusion, which is reflected in the article's title, remains valid. Please see below for specific comments.\nWe sincerely thank the reviewer for the time, care, and thoughtful attention devoted to evaluating both Reviewer #1’s comments and our responses. We truly appreciate the acknowledgement of the efforts we made to address the points raised, particularly regarding vesicle heterogeneity and reconstitution limitations. In response, we have added a short paragraph in the Discussion to explicitly acknowledge this limitation while clarifying how it is mitigated:\n“…Finally, while liposome size control after reconstitution is inherently limited, the applied protocols reliably generate predominantly unilamellar vesicles (DOI:10.1038/nprot.2007.519). Importantly, the assay design–using very low transporter-to-lipid ratios and orientation-specific surface immobilization– selectively removes empty, oversized, and non-functional liposomes, resulting in a highly enriched population of function proteoliposomes with an apparent size of ~100 nm (Supplementary Fig. 11).\nWe hope this addition addresses the concern and further strengthens the clarity and rigor of our manuscript.\nReply to Reviewer Response Point 9 and 12:\nLiposomes were prepared by thin-film hydration, followed by sonication, five freeze-thaw cycles, and extrusion through a 100 nm polycarbonate membrane prior to reconstitution. Freeze-thaw cycling is known to substantially reduce multi-lamellarity, yielding approximately 91% unilamellar vesicles, while extrusion further disrupts the remaining multilamellar structures, as demonstrated previously (DOI: 10.1021/acs.langmuir.8b04256;DOI: 10.1021/acs.langmuir.8b04256; DOI: 10.1016/s0378-5173(01)00721- 9; DOI:10.1016/0378-5173(87)90139-6). In addition, protein reconstitution was performed following the well-established protocol by Geertsma et al. (2008), which is reported to produce unilamellar proteoliposomes with a low fraction of multilamellar vesicles (DOI: 10.1038/nprot.2007.519.).\nWe acknowledge that the multi-lamellarity is not directly probed by the fluorescence readout itself. However, the assay design strongly disfavors contributions from multilamellar vesicles. Following extrusion through pores <200 nm, only ~9% of bilamellar vesicles are expected to remain. Moreover, the experimental configuration requires that OppA be encapsulated within the same vesicle as TmrAB for transport activity to be detected. Multilamellar vesicles, incorrectly oriented transporters, or liposomes lacking functional encapsulation of OppA are therefore intrinsically excluded from the functional and single-molecule analyses. Additionally, non-tethered liposomes were removed by washing, further enriching for assay-competent vesicles.\nRegarding liposome size, nanoparticle tracking analysis (NTA) was performed on samples directly after reconstitution, a method known to overestimate the contribution of larger particles and to broaden the apparent size distribution. In contrast, samples used for single-molecule FRET measurements underwent an additional centrifugation step (2,000 × g, 2 min) prior to surface immobilization. This step efficiently removes large and aggregated liposomes, explaining the narrower size distribution observed under single-molecule conditions. Furthermore, highly fluorescent aggregates (e.g., the bright spot visible in Supplementary Fig. S11) were excluded from analysis based on DiD membrane staining. As a result, the liposome population analyzed at the surface is effectively enriched for ~100-nm vesicles.\nTaken together, the photobleaching statistics, established liposome preparation protocols, and the intrinsic selectivity of the assay design collectively support the conclusion that the analyzed proteoliposomes predominantly contain a single transporter embedded in unilamellar vesicles.\nReviewer Response to Point 13: The authors have done the requested experiment. I believe that the majority of the vesicles with TmrAB have 1 transport protein per liposome, however it is hard to believe that the accuracy is 96.5%.\nThe reported value of 95.6% was calculated based on 2,088 individual photobleaching traces. Of these, 1,997 traces (95.6%) displayed a single-step photobleaching event, consistent with the presence of a single transporter per liposome, while only 91 traces (4.4%) showed a two-step photobleaching event, indicative of two transporters per liposome (Supplementary Fig.6). These results are in good agreement with the chosen reconstitution conditions, which were designed to yield, on average, one transporter per ten liposomes, resulting in a population dominated by empty liposomes and a low probability of multiple transporters per vesicle. We therefore conclude that the vast majority (>90%) of transporter-containing liposomes harbor only a single reconstituted transporter."}, "author_response": ""}]

Decision letter