{"paper_id":"3c340c4a-0184-4fe8-8776-b9d5f018ceed","body_text":"1 \nATP-driven conformational dynamics reveal hidden intermediates  1 \nin a heterodimeric ABC transporter 2 \nMatija Pečak1, Christoph Nocker1, Robert Tampé1* 3 \n1 Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Max-von-Laue Str. 9, 4 \nFrankfurt a.M., Germany 5 \n*Corresponding author: tampe@em.uni-frankfurt.de 6 \nAbstract 7 \nATP-binding cassette (ABC) transporters are essential molecular machines whose conformational 8 \ndynamics have largely been inferred from ensemble-averaged measurements . Resolving dynamic 9 \nheterogeneity and transient intermediates, however, requires single-molecule approaches. Here, we 10 \nuse single-molecule Förster resonance energy transfer (smFRET) to directly monitor conformational 11 \nchanges of the heterodimeric type IV ABC transporter TmrAB, a functional homolog of the human 12 \nantigen transporter TAP . Fluorophores positioned at the nucleotide-binding domains and the 13 \nperiplasmic gate were validated by accessible-volume simulations, fluorescen ce lifetimes, and 14 \nensemble FRET, demonstrating that these reporters reliably track conformational transitions . Single-15 \nmolecule analysis distinguishes ATP-free and ATP-bound states and reveals ATP-dependent 16 \npopulation shifts from nucleotide-free to physiological ATP concentrations . Probing conformational 17 \ndwell-times further uncovers an unexpectedly long ATP-bound dwell time of ~300 ms. Using 18 \ncomplementary stabilization strategies –including a slow-turnover variant, Mg²⁺ depletion, o r 19 \nsubstrate trans-inhibition –we resolve a previously hidden outward-facing open state that rapidly 20 \ninterconverts with occluded intermediates under turnover conditions. These results provide the first 21 \nsingle-molecule characterization of TmrAB and establish a general framework for dissecting ATP-22 \ncoupled conformational dynamics in heterodimeric ABC transporters. 23 \n 24 \nKeywords: ABC transporters, conformational dynamics, membrane protein, single-molecule analysis  25 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n2 \nIntroduction 26 \nATP-binding cassette (ABC) transporters constitute the largest family of primary active membrane 27 \ntransport systems, conserved across all domains of life1-3. Despite considerable structural diversity, all 28 \nABC transporters share a modular architecture compris ing two conserved nucleotide-binding 29 \ndomains (NBDs)–the defining hallmark of the family –and two transmembrane domains (TMDs) that 30 \nform the substrate translocation pathway 3,4. Based on their transmembrane-domain architecture , 31 \nABC transporters are classified into seven types that encompass importers, exporters, extractors, and 32 \nmechanotransmitters5. Substrate translocation is driven by large conformational changes that are 33 \nchemo-mechanically coupled to ATP binding, hydrolysis, and phosphate/ADP release 2,3. ABC 34 \ntransporters play central roles in cellular homeostasis, nutrient uptake, waste removal, and toxin 35 \ndefense. Their dysfunction and misregulation are linked to numerous diseases and drug resistance6. 36 \nThe heterodimeric type IV ABC transporter TmrAB from Thermus thermophilus  has emerged as a 37 \npowerful model system due to its exceptional thermal stability and functional homology to the 38 \ntransporter associated with antigen processing (TAP1/2), a key component of adaptive immunity 7-9. 39 \nNotably, TmrAB shares overlapping peptide specificity with TAP and can restore antigen presentation 40 \nin TAP-deficient human cells 10. Its inherent asymmetry, with one catalytically active (canonical) and 41 \none inactive (noncanonical) nucleotide-binding site (NBS), provides a unique opportunity to 42 \ninvestigate functional specialization and asymmetry in ABC transport mechanisms.  43 \nExtensive structural studies, particularly using cryogenic electron microscopy (cryo-EM), ha ve 44 \ndelineated the conformation al landscape of TmrAB and yielded a detailed model of its translocation 45 \ncycle11,12. In this model, TmrAB fluctuates between inward-facing wide and narrow conformations 46 \n(IFwide and IFnarrow), characterized by a sealed periplasmic gate (PG) and well-separated NBDs, thereby 47 \npermitting substrate access to the central binding cavity. ATP binding to both NBDs induces NBD 48 \ndimerization and drives the transition into the outward-facing states, including an OF open (OF open) 49 \nconformation with an open PG that enables substrate release into the periplasm, as well as an OF 50 \noccluded (OF occluded) state characterized by a sealed PG and dimerized NBDs. Subsequent ATP 51 \nhydrolysis and phosphate release lead to asymmetric unlocked return states (UR asym and URasym*), 52 \nbefore the transporter returns to the IF conformation. These UR sates feature as sealed PG, a 53 \npartially open ADP-bound canonical NBS, and a tightly ATP-occluded noncanonical NBS11.  54 \nSingle-turnover experiments established that ATP binding, rather than hydrolysis, drives the IF-to-OF 55 \ntransition, while phosphate release precedes the OF-to-IF switch 12,13. Complementary ensemble 56 \napproaches, including pulsed electron –electron double resonance (PELDOR/DEER) spectroscopy, 57 \nhave further characterized ATP-dependent conformational changes 14,15. Howe ver, ensemble 58 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n3 \naveraging inherently masks molecular heterogeneity, obscures inactive or misfolded subpopulations, 59 \nand limits access to kinetic information.  60 \nSingle-molecule techniques overcome these limitations by resolving conformational dynamics at the 61 \nlevel of individual molecules 16,17. In particular, single-molecule Förster resonance energy transfer 62 \n(smFRET) enables real-time monitoring of protein conformational changes with nanometer 63 \nprecision18-21. Applied to ABC transporters, smFRET provides a unique opportunity to dissect 64 \ntransport cycles,  resolve transient intermediates, and extract kinetic and mechanistic insights that 65 \nremain inaccessible to ensemble-based measurement approaches22-24. 66 \nHere, we apply total internal reflection fluorescence (TIRF) microscopy combined with alternating 67 \nlaser excitation (ALEX)-based smFRET to detergent-solubilized heterodimeric ABC transporter  TmrAB, 68 \nproviding the first single-molecule characterization of this system. By strategically positioning 69 \nfluorophore pairs, we directly monitor ATP-dependent NBD dimerization and periplasmic gate ( PG) 70 \nopening, quantify conformational state occupancies across ATP concentrations ranging from 71 \nnucleotide-free to physiological levels (3 mM), and uncover conformational dynamics previously 72 \nmasked by ensemble averaging. Using three orthogonal trapping strategies –(i) a slow-turnover 73 \ncatalytic mutant 11,12, (ii) Mg²⁺ depletion 14,25, and (iii) substrate trans-inhibition 26,27–we resolved a 74 \npreviously hidden outward-facing open (OF open) state that rapidly exchanges with the outward-facing 75 \noccluded (OFoccluded) state. Distance measurements derived from smFRET closely match ed predictions 76 \nfrom accessible-volume (AV) simulations, cryo-EM structures, and PELDOR/DEER spectroscopy, 77 \nconfirming that detergent-solubilized TmrAB retains a native-like conformational landscape. 78 \nTogether, these results provide the first single-molecule quantification of conformational state 79 \noccupancies for a heterodimeric type IV ABC transporter and establish TmrAB as a versatile model 80 \nfor single-molecule studies of ABC transport systems.  81 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n4 \nResults 82 \nDesign of FRET-labeled TmrAB variants to probe conformational dynamics 83 \nTo monitor conformational changes in distinct regions of TmrAB, w e engineered FRET variants 84 \ntargeting the nucleotide-binding domains (NBDs) and the periplasmic gate (PG). The NBDs undergo 85 \nATP-dependent dimerization followed by post-hydrolysis dissociation , whereas the PG opening and 86 \nclosing controls substrate release into the periplasm 2,3,11. Probing both regions provides 87 \ncomplementary readouts of cytosolic and periplasmic coupling during the transport cycle.  88 \nLabeling positions were selected based on prior PELDOR/DEER studies 15. The NBD report er variant 89 \n(TmrAC416BL458C, referred to as TmrAB NBD) monitors conformational changes at the noncanonical 90 \nnucleotide-binding site (NBS) , while the PG reporter (TmrA C416A, T61CBR56C, hereafter TmrAB PG) tracks 91 \nPG opening. In TmrAB NBD, the native single cysteine (C416) was retained for labeling, whereas in 92 \nTmrABPG it was substituted by alanine to prevent off-target labeling. Selecting the noncanonical 93 \nrather than the canonical NBS prevents distinguish ing outward-facing occluded (OF occluded) from 94 \nasymmetric unlocked return states (URasym and URasym*)11, but reduces the number of resolvable FRET 95 \nstates and thereby simplifies data interpretation.  96 \nBoth variants were labeled with photostable fluorophores, LD555 (donor) and LD655 (acceptor), 97 \ncontaining a 1,3,5,7-cyclooctatetraene moiety to suppress photobleaching and blinking 28,29. 98 \nAccessible-volume (AV) simulations 30 performed on nine cryo-EM structures11 confirmed that donor-99 \nacceptor distances ( RDA) and simulated FRET efficiencies ( Esim) fall within the FRET-sensitive range 100 \n(Fig. 1). For TmrAB NBD, Esim ranged from 0.62 ± 0.02 (57.9 ± 0.7 Å, NBDs separated) to 0.84 ± 0.01 101 \n(45.2 ± 0.4 Å, NBDs dimerized). For TmrAB PG, Esim shifted from 0.96 ± 0.02 (30.6 ± 0.4 Å, PG closed) to 102 \n0.69 ± 0.03 (53.8 ± 2.0 Å, PG open). These transitions correspond to ΔEsim values of 0.22 and 0.27 and 103 \nΔRDA of 12.7 Å and 23.2 Å, for TmrAB NBD and TmrAB PG, respectively, predicting robust and 104 \nexperimentally resolvable FRET changes. 105 \nAdditionally, we employed a slow-turnover TmrAB variant that reports on PG opening (TmrA C416A, 106 \nE523Q, T61C BR56C, hereafter TmrAB PG_EQ). Substituting the catalytic glutamate with glutamine removed 107 \nthe carboxylate required to activate water for nucleophilic attack on ATP, thus drastically reducing 108 \nthe rate of ATP hydrolysis. This mutation slows down the catalytic turnover (~1000-fold) to a half-life 109 \nof approximately 25 min at 45 ˚C11,12,14,25, enabling stabilization of ATP-bound outward-facing 110 \nconformations.  111 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n5 \nTmrAB variants are suitable for FRET studies 112 \nTmrAB variants were expressed in E. coli and purified using immobilized metal-affinity 113 \nchromatography. SDS-PAGE and size-exclusion chromatography (SEC) confirmed high sample purity 114 \nand monodispersity (Fig. 1–Fig. S1a,b). ATPase assays of TmrABwt verified that enzymatic activity was 115 \nfully retained after purification, yielding a Michaelis-Menten constant ( Km) of 0.97 ± 0.28 mM and 116 \ncatalytic ATP turnover rate (kcat) of 2.57 ± 0.38 s-1 at 40 ˚C (Fig. 1–Fig. S1c). 117 \nCysteine-maleimide labeling of detergent-solubilized TmrAB variants achieved site-specific labeling 118 \nefficiencies exceeding 90% ( Fig. 1–Fig. S1d–f). Fluorescence lifetime ( τ) analysis of conjugated 119 \nfluorophores confirmed that their photophysical properties were preserved and that they retained 120 \nsufficient rotational freedom for reliable FRET measurements. τ histograms of both conjugated and 121 \nfree fluorophores were fitted with a biexponential decay model, from which amplitude-weighted 122 \naverage lifetimes were calculated. For TmrAB NBD, average τ values were 0.93 ± 0.02 ns (LD555) and 123 \n1.52 ± 0.01 ns (LD655), while TmrAB PG exhibited average τ values of 0.95 ± 0.02 ns (LD555) and 124 \n1.65 ± 0.01 ns (LD655) ( Fig. 1–Fig. S2a). By comparison, free dyes in buffer displayed lifetimes of 125 \n1.11 ± 0.02 ns (LD555) and 1.29 ±0.01 ns (LD655). Because the fluorescence lifetimes of both, the 126 \nconjugated dyes and the free dyes, remain on the ~1 ns timescale, we conclude that the 127 \nfluorophores remain photophysically active and are not affected by protein-induced quenching 17,31. 128 \nMoreover, the measured lifetimes on the nanosecond timescale are only marginally affected and, 129 \nmost importantly, identical between the TmrAB variants, indicating dynamics orientational averaging 130 \nof the transition dipoles and confirming that the labeled constructs are suitable for quantitative FRET 131 \nstudies32-34. 132 \nEnsemble ATP titration (0–10 mM ATP) revealed the expected concentration-dependent donor 133 \nquenching and acceptor sensitization ( Fig. 1–Fig. S2b–d). ATP-induced fractional fluorescence 134 \nchanges provided as a quantitative readout of conformational transitions, allowing estimation of 135 \nequilibrium dissociation constants for ATP binding (Kd, ATP) to labeled TmrAB variants ( Fig. 1–Fig. S2e–136 \ng). The measured apparent Kd, ATP values––51 ± 38 μM for TmrAB NBD, 68 ± 25 μM for TmrAB PG, and 137 \n95 ± 26 μM for the slow -turnover variant TmrAB PG_EQ––are in good agreement with previously 138 \nreported values (~100 µM for TmrA E523QB)12, indicating that fluorophore labeling does not perturb 139 \nATP binding . Notably, TmrAB PG_EQ exhibited a larger shift in ATP-induced fluorescence change than 140 \nTmrABPG, consistent with stabilization of the ATP-bound conformation and reduced catalytic 141 \nturnover. 142 \n 143 \n 144 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n6 \n 145 \nATP-induced conformational switching resolved by single-molecule FRET 146 \nTmrAB variants were site-specifically immobilized on PEGylated coverslips using a conformation-147 \nindependent, TmrB-specific nanobody ( Nb9F10S63C)11 conjugated to maleimide-PEG 11-biotin (Fig. 2a). 148 \nPrevious studies confirmed that this nanobody does not perturb TmrAB transport or ATPase 149 \nactivity11,25. Donor and acceptor photons were recorded by a total-internal reflection fluorescence 150 \n(TIRF) microscope using alternating laser excitation (ALEX; NanoImager) at 40 ˚C ( Fig. 2b). Single-151 \nmolecule localization , fluorescence-trajectory extraction, and background correction were 152 \nperformed using NanoImager software, followed by DeepFRET-based machine-learning trace 153 \nclassification and corrections for donor leakage, direct acceptor excitation, and difference in 154 \ndetection-efficiency17,35,36 ( Fig. 2–Fig. S1 and 2). FRET efficiency ( E) and stoichiometry ( S) were 155 \ncalculated from the corrected fluorescence-trajectories (see Methods, Eq. 1 and Eq. 2). 156 \nFRET efficiency (E) histograms revealed two Gaussian populations corresponding to the apo and ATP-157 \nbound states ( Fig. 2d,e). In the absence of ATP, only the apo population was observed, whereas 158 \naddition of 3 mM ATP induced the appearance of a second ATP-bound population. For TmrAB NBD, the 159 \napo and ATP-bound populations exhibited mean E values of 0.58 and 0.88 ( ΔE = 0.30), respectively, 160 \nwith ~77% of molecules occupying the ATP-bound state. For TmrAB PG, mean E values were 0.97 (apo) 161 \nand 0.86 (ATP-bound) (ΔE = 0.11), with ~80% of molecules in the ATP-bound state.  162 \nDistance estimates calculated using a Förster radius of R0 = 63.5 Å (ref.37) yielded apparent distances 163 \nof 60.2 Å (apo) and 45.6 Å (ATP-bound) for TmrAB NBD, and 35.6 Å (apo) and 46.9 Å (ATP-bound) for 164 \nTmrABPG. For TmrAB NBD, the experimentally derived ΔR of 14.6 Å closely agreed with the AV 165 \nsimulations. In contrast , the smaller Δ R of 11.4 Å observed for TmrAB PG deviated from simulated 166 \nvalues, indicating that the ATP-bound population at this site represents a mixture of rapidly 167 \ninterconverting conformations rather than a single well-defined state. 168 \nTo assess the ATP sensitivity, we quantified conformational responses across a wide range of ATP 169 \nconcentrations, spanning well below the reported Kd, ATP (~ 100 µM for TmrA E523QB)12 up to 170 \nphysiologically relevant levels (3 mM ATP) . smFRET measurements revealed dose-dependent 171 \npopulation shifts: TmrAB NBD transitioned from a low-FRET apo state ( E = 0.58) to high-FRET ATP-172 \nbound state ( E = 0.88), whereas TmrAB PG shifted from a high-FRET apo state ( E = 0.97) to a lower-173 \nFRET ATP-bound state ( E = 0.86) (Fig. 3a,c). Langmuir isotherm fits yielded Kd, ATP values of 13 ± 1 μM 174 \nfor TmrAB NBD and 2 ± 1 μM for TmrAB PG (Fig. 3b,d), indicating saturation at ATP concentrations well 175 \nbelow physiological levels (3 mM). For the TmrAB PG variant, population quantification and 176 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n7 \nsubsequent determination of Kd, ATP are unreliable due to insufficient separation of the two FRET 177 \npopulations below 1 mM ATP, consistent with the smaller ΔE observed for this labeling configuration.  178 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n8 \nTrapping of TmrABPG reveals a previously hidden outward-facing open state 179 \nAt physiological ATP concentrations (3 mM), ~80% of TmrAB PG molecules populated the ATP-bound 180 \nstate (E = 0.86), closely matching the ATP-bound population (~77% of NBD-dimerized) observed for 181 \nTmrABNBD ( Fig. 3). This agreement indicates that both labeling strategies consistently report ATP-182 \ndependent conformational changes. However, the ATP-induced shift observed for the periplasmic-183 \ngate reporter TmrABPG (ΔE = 0.11, ΔR = 11.4 Å; Fig. 3c) was substantially smaller than predicted by AV 184 \nsimulations ( ΔE = 0.27, ΔR = 23.2 Å; Fig. 1c). This discrepancy indicates that the ATP-bound 185 \npopulation at E = 0.86 represents an unresolved ensemble, potentially comprising OFopen and 186 \nOFoccluded conformations, as well as the post-hydrolysis asymmetric unlocked return states (UR asym and 187 \nURasym*), which are clearly indistinguishable from OFoccluded within the current FRET geometry11.  188 \nTo test whether these states are kinetically unresolved by smFRET , we applied three complementary 189 \nstrategies to arrest the OFopen conformation of TmrAB PG: (i) a slow-turnover catalytic mutant 190 \n(TmrABPG_EQ), (ii) Mg²⁺ depletion using EDTA, and (iii) reverse inhibition by high concentrations of the 191 \nsubstrate peptide RRYQKSTEL (R9L) ( Fig. 4). Slow-turnover variants have previously enabled 192 \nstructural separation of OFopen and OF occluded states 11,12,14,25. Mg²⁺ depletion blocks ATP hydrolysis 193 \nwhile preserving ATP binding, allowing rapid and reversible trapping 25. We further hypothesized that 194 \ntrans-inhibition by peptide binding sterically restricts PG closure and is therefore expected to 195 \nstabilize OFopen in a dose-dependent manner26,27.  196 \nIn conditions lacking ATP, either in the absence of nucleotides or in the presence of ADP (3 mM ADP), 197 \nthe slow-turnover variant TmrAB PG_EQ populated a single high-FRET state ( E = 0.97) (Fig. 4a, top and 198 \nmiddle). These results indicate that ADP binding alone is insufficient to promote NBD dimerization 199 \nnor PG opening, consistent with previous biochemical and structural observations 11,12. Upon ATP 200 \naddition (3 mM ATP), however, the conformational landscape diverged sharply from that of wild-201 \ntype TmrABPG. Instead of the two-state distribution observed for wild-type (apo : E = 0.97, ~20%; ATP-202 \nbound: E = 0.86, ~80%) (Fig. 2e, bottom), TmrAB PG_EQ exhibited three well-resolved populations with 203 \nmean E values of E = 0.97 (~14%), E = 0.86 (~55%), and E = 0.63 (~31%) (Fig. 4a, bottom).  204 \nAs in wild-type TmrABPG, the high-FRET population (E = 0.97) corresponds to the IF state, whereas the 205 \nintermediate-FRET population (E  = 0.86) likely represents a dynamic equilibrium of OF open and 206 \nOFoccluded conformations, as suggested by cryo-EM analyses 11. Post-hydrolysis return states ( URasym 207 \nand URasym*) are expected to be minimally populated in TmrAB PG_EQ due to its drastically reduced ATP 208 \nhydrolysis rate12. Notably, the low-FRET ATP-bound population (E = 0.63) was entirely absent in wild-209 \ntype. The transition from E = 0.97 to E = 0.63 corresponds to ΔE = 0.34 and ΔR = 22.5 Å, in close 210 \nagreement with the IF OFopen distance predicted by AV simulations ( ΔR = 23.2 Å; Fig. 1), thereby 211 \npostulating E = 0.63 as the OFopen conformation. 212 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n9 \nMg²⁺ depletion independently reproduced this three-state landscape. In the absence of Mg 2+, wild-213 \ntype TmrABPG transitioned from a single apo population (without ATP; E = 0.97) (Fig. 4b, top) to three 214 \nATP-bound populations (3 mM ATP; E = 0.97, ~13%; E = 0.86, ~56%; E = 0.63, ~31%) ( Fig. 4b, middle) 215 \nclosely resembling those observed for the slow-turnover variant ( Fig. 4a, bottom) . Reintroducing 216 \nMg²⁺ abolished the E = 0.63 population and restored the wild-type two-state distribution ( Fig. 4b, 217 \nbottom). This reversibility confirmed that the ATP-bound OFopen state (E = 0.63) is selectively revealed 218 \nonly when ATP hydrolysis is prevented. 219 \nFinally, we tested whether periplasmic substrate binding shifts the conformational equilibrium of 220 \nwild-type TmrAB PG toward OFopen. In the presence of ATP (3 mM), increasing concentrations of 221 \npeptide substrate (0.3–2 mM R9L) progressively enriched the E = 0.63 population from ~20% to ~38% 222 \n(Fig. 4c). This dose-dependent stabilization mirrors trans-inhibition behavior reported for human 223 \nTAP1/2 and reflects the upper substrate-loading capacity of the transporter26.  224 \nDistance changes derived from smFRET closely match AV simulations, cryo-EM structures (PDB 6RAH, 225 \n6RAN)11, and DEER/PELDOR measurements 14 (Fig. 4d), together validating assignment of the E = 0.63 226 \npopulation as the OFopen conformation. 227 \n 228 \nKinetics and thermodynamics of the transport cycle  229 \nALEX-smFRET data were acquired with an effective temporal resolution of 2 00 ms (100 ms per 230 \nexcitation channel). Shorter integration times compromised the signal-to -noise ratio and precluded 231 \nreliable FRET determination . To quantify conformational dynamics, we applied Hidden Markov 232 \nModeling (HMM) using MASH-FRET 38, classifying traces as either static (single FRET state) or dynamic 233 \n(multiple states). Approximately 95% of traces in each condition were classified as static, indicating 234 \nthat most conformational transitions occur at or below our temporal resolution.  235 \nAlthough individual transitions could not be directly resolved, population-based analysis ( Fig. 3), 236 \ncombined with biochemical turnover measurements ( Fig. 1–Fig. S1c), allowed estimation of ATP-237 \nbound dwell times. At saturating ATP conditions well above the apparent Kd, ATP (3 mM, 40 ˚C), wild-238 \ntype TmrAB exhibited a catalytic turnover rate of kcat = 2.57 ± 0.38 s-1 (Fig. 1–Fig. S1c), corresponding 239 \nto a full transport cycle time ( τcycle) of 395 ± 55 ms. ATP-bound dwell times ( τd) were derived from 240 \npopulation ratios obtained from Gaussian fits of the FRET efficiency histograms ( Fig. 3; see Methods, 241 \nEq. 3 and Eq. 4). These analyses yielded ATP-bound dwell times of 304 ± 43 ms for TmrAB NBD (~77% 242 \nATP-bound) and 316 ± 44 ms for TmrAB PG (~80% ATP-bound), with the remaining ATP-free intervals 243 \n(~20–23%) accounting for 91 ms and 79 ms of the cycle, respectively. 244 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n10 \nTogether, these measurements establish a quantitative, single-molecule description of conformation 245 \nstate occupancies and dwell times throughout the catalytic cycle of a heterodimeric ABC transporter 246 \nunder active turnover conditions, providing valuable insights into the dynamic landscape of its 247 \ntranslocation cycle. 248 \n 249 \nDiscussion 250 \nsmFRET has become an indispensable tool for dissecting conformational dynamics of membrane 251 \nproteins, including receptors, ion channels, and transporters, by directly linking structural transitions 252 \nto functional states With the ABC transporter family, however, smFRET studies have largely been 253 \nconfined to monomeric or homodimeric systems 22-24, leaving the dynamic behavior of asymmetric, 254 \nheterodimeric transporter comparatively unexplored. Here, we apply smFRET to the heterodimeric 255 \ntype IV ABC transporter TmrAB, extending single-molecule analysis to an asymmetric transporter 256 \nsystem and uncovering dynamic features of the transport cycle that are inaccessible to ensemble-257 \naveraged approaches. 258 \nBy positioning FRET reporters at the nucleotide-binding domains (NBDs) and periplasmic gate (PG), 259 \nwe directly monitored ATP-dependent coupling between chemical energy input and global 260 \nconformational rearrangements. Importantly, these structural rearrangements are not strictly 261 \ncorrelated: NBD dimerization can give rise to either an outward-facing open (OF open) or occluded 262 \n(OFoccluded) conformation11,12. This decoupling underscores the need to monitor both cytosolic and 263 \nperiplasmic regions to resolve the transport mechanism.  264 \nLabeling sites previously validated for PELDOR/DEER spectroscopy 10,14,15 were adapted for smFRET 265 \nand rigorously benchmarked using accessible-volume (AV) simulations 30, fluorescence lifetime 266 \nanalysis, and ensemble FRET titrations. This additional validation is essential because fluorophores 267 \nimpose stricter steric and rotational constraints than nitroxide spin labels 32. Collectively, these 268 \ncontrols demonstrate that fluorophore attachment preserves native-like conformational behavior 269 \nand ATP binding, establishing TmrAB as a robust system for quantitative single-molecule analysis. 270 \nConsistent with this conclusion, fluorescence lifetime analysis showed no evidence for substantial 271 \nprotein-fluorophore quenching or restricted dye motion, as indicated by donor lifetime shortening 272 \nand prolonged acceptor lifetimes characteristic of efficient energy transfer17.  273 \nSingle-molecule measurements resolved two dominant FRET populations corresponding to apo and 274 \nATP-bound states for both reporter variants TmrAB NBD and TmrAB PG. ATP titrations spanning 275 \nconcentrations well below the reported apparent Kd, ATP (~100 µM for TmrA E523QB)12 up to 276 \nphysiologically relevant levels (3 mM ATP) revealed gradual, concentration-dependent shifts 277 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n11 \nbetween these populations, demonstrating the high sensitivity of the FRET constructs to ATP binding. 278 \nNotably, apparent Kd, ATP values derived from smFRET (2 –13 µM) were substantially lower than those 279 \nobtained from ensemble FRET measurements (50 –100 µM). This difference likely reflects the 280 \ninherent selectively of single-molecule analyses for properly folded and catalytically competent 281 \ntransporters. In smFRET experiments, aggregated or inactive species can be identified and excluded 282 \nduring trace selection based on fluorescence intensity, stoichiometry, and photobleaching behavior, 283 \nthereby enriching the analyzed population for functional molecules. 284 \nCrucially, the ATP-bound population may reflect a rapidly interconverting ensemble of 285 \nconformations, rather than a single static structure, potentially including OFopen, OFoccluded, and post-286 \nhydrolysis unlocked return states (UR asym and UR asym*). These transitions occur faster than the 287 \n~200 ms temporal resolution of our measurements, resulting in averaged FRET efficiencies under 288 \nturnover conditions. Using three independent trapping strategies —slow-turnover catalysis, Mg²⁺ 289 \ndepletion, and substrate trans-inhibition— we stabilized and directly resolved a previously hidden 290 \nOFopen conformation. The associated distance changes are generally consistent with cryo-EM 291 \nstructures11, PELDOR/DEER data 10,14,15, and simulation-based predictions, suggesting that detergent-292 \nsolubilized TmrAB samples a largely native-like conformational landscape. 293 \nAlthough detergent-solubilized and lipid nanodisc-reconstituted TmrAB exhibit similar global 294 \nconformational states, the conformational space accessible to attached fluorophores may be 295 \ndifferentially influenced by membrane-associated environments 19,39,40. In particular, fluorophores 296 \nattached near the periplasmic gate may experience steric restrictions due to partial overlap with the 297 \nmembrane region, as suggested by AV simulations. While such effects are negligible under the 298 \ndetergent conditions used here , they should be carefully evaluated in future studies employing 299 \nmembrane-embedded systems. Single-molecule measurements further revealed that addition of the 300 \npeptide substrate induces concentration-dependent shifts in the conformational equilibrium. 301 \nIncreasing substrate concentrations progressively stabilized the OF open state, consistent with trans-302 \ninhibition behavior observed in human TAP 1/226 and bovine ABCC1 27, and reflecting the finite 303 \nsubstrate-loading capacity of the transporter26.  304 \nQuantitative deconvolution of FRET populations enabled direct determination of conformational 305 \nstate occupancies under physiological ATP concentrations. During active turnover, TmrAB populates 306 \nthe IF state (~20%), the OFopen state (~25%), and OF occluded/post-hydrolysis states (UR asym and URasym*) 307 \n(~55%) (Fig. 5). The current reporter geometries do not allow direct discrimination between OFoccluded 308 \nand post-hydrolysis states because fluorophores were placed at the noncanonical nucleotide-binding 309 \nsite. However, contributions from post-hydrolysis states are expected to be minimal for slow-310 \nturnover TmrAB variant (TmrAB PG_EQ), owing to its drastically reduced ATP hydrolysis rate. To our 311 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n12 \nknowledge, this represents the first single-molecule quantification of conformational equilibria for a 312 \nheterodimeric ABC transporter under catalytic conditions. While these distributions broadly align 313 \nwith cryo-EM particle classifications 11,25, smFRET resolves fewer IF states than the cryo-EM  314 \ndistinction between IF wide and IF narrow, and additionally captures ATP-bound intermediates that are 315 \nchallenging to resolve structurally due to rapid interconversion.  316 \nAll smFRET measurements were performed at 40 ˚C to maintain consistency with prior biochemical 317 \nstudies and ensure fluorophore stability. At the physiological temperature of T. thermophilus (68 ˚C), 318 \nabsolute rates of ATP turnover and conformational transitions are expected to increase, although 319 \nrelative state occupancies may remain conserved if the underlying free-energy landscape is 320 \npreserved. Despite a substantial fraction of static single-molecule trajectories, ATP-dependent 321 \npopulation shifts and catalytic rates indicate that TmrAB operates near the temporal resolution limit 322 \nof our measurements. Integrating smFRET-derived state occupancies with biochemical turnover rates 323 \nyields an ATP-bound dwell time of approximately 300 ms, in good agreement with previous 324 \nbiochemical estimates9,12.  325 \nEmerging microsecond-resolution smFRET approaches offer the potential to direct ly visualize short-326 \nlived intermediates within the transport cycle 41. Future studies could further benefit from three- or 327 \nfour-color FRET strategies 42,43, which would allow simultaneous monitoring of multiple structural 328 \nelements. In particular, dual labeling of the NBDs and PG could provide direct detection of the 329 \nOFoccluded state, while probes placed at both canonical and noncanonical nucleotide-binding sites 330 \ncould capture post-hydrolysis conformational dynamics. 331 \nIn summary , this work establish es smFRET as a powerful approach for mapping the dynamic 332 \nlandscape of asymmetric ABC transporters. By quantitatively linking ATP binding, conformational 333 \nequilibria, and kinetics at the single -molecule level, our study resolved an important aspect of the 334 \ntransport mechanism how chemical energy is transduced into directional transport in heterodimeric 335 \nABC systems. Integration of native lipid environments, higher temporal resolution, and substrate 336 \nengagement will further illuminate the coordination of ATP hydrolysis and substrate translocation 337 \nduring transport.  338 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n13 \nMethods 339 \nExpression, purification, and labeling of TmrAB 340 \nHis10-tagged TmrAB variants were expressed in E. coli BL21(DE3) (Thermo Fisher Scientific) as 341 \ndescribed previously 12. Cells were grown in high-salt LB media (Carl Roth) supplemented with 342 \n100 μg ml-1 ampicillin (PAA Laboratories) at 37 ˚C. At an OD 600 of 0.5, expression was induced with 343 \n1 mM isopropyl β -D-thiogalactopyranoside (IPTG; Carl Roth), and cultures were incubated for 3 h at 344 \n37 ˚C. Cells were harvested by centrifugation (4,500 × g, 4 ˚C, 15 min) and stored at –80 ˚C.  345 \nFor purification, cell pellets were resuspended in lysis buffer (20 mM HEPES-NaOH pH 7.5, 300 mM 346 \nNaCl, 50 µg ml-1 lysozyme, 0.2 mM phenylmethylsulfonyl fluoride (PMSF)) and lysed by sonication. 347 \nCell debris was removed by centrifugation (18,000 × g, 4 ˚C, 35 min), and membranes were collected 348 \nby ultracentrifugation (100,000 × g, 4 ˚C, 30  min). Membranes were solubilized for 2 h at 4 ˚C in 349 \npurification buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl) containing 20 mM n- dodecyl β-D-350 \nmaltoside (β-DDM; Carl Roth). After ultracentrifugation (100,000 × g, 30 min, 4 ˚C), the supernatant 351 \nwas incubated with Ni- NTA agarose (Bio-Rad) for 1 h at 4 ˚C. The resin was washed with 20 column 352 \nvolumes of wash buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 1 mM β -DDM) containing 353 \n50 mM imidazole, and TmrAB was eluted with elution buffer (20 mM HEPES-NaOH pH 7.5, 300 mM 354 \nNaCl, 1 mM β-DDM, 300 mM imidazole).  355 \nFor fluorophore labeling, TmrAB variants were conjugated via  maleimide chemistry using LD555 and 356 \nLD655 (Lumidyne Technologies). Labeling was carried out at a 1:10:10 molar ratio of protein to each 357 \ndye in elution buffer for 3 h at 4 ˚C. Excess dye was quenched with 2 mM β-mercaptoethanol (Sigma-358 \nAldrich), and the labeled protein was buffer-exchanged into size-exclusion chromatography (SEC) 359 \nbuffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM β -DDM) using a Zeba Spin Desalting 360 \nColumn (Thermo Fisher Scientific). Unreacted fluorophores were removed by SEC on a Superdex 200 361 \nIncrease 10/300 GL column (Cytiva). Labeling efficiency was determined by analytical SEC (Superdex 362 \n200 Increase 3.2/300; Cytiva) by monitoring absorbance at 280, 555, and 655 nm. To preserve 363 \nsample integrity for smFRET measurements, TmrAB was purified and labeled within a single day, 364 \nstored on ice, and imaged over the following two days. 365 \n 366 \nTime-correlated single-photon counting (TCSPC) 367 \nFluorescence lifetime measurements were performed using a Fluo Time 100 spectrometer 368 \n(PicoQuant) equipped for time-correlated single-photon counting (TCSPC). Experiments were carried 369 \nout on labeled TmrAB in SEC buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM β -DDM). 370 \nLD555 and LD655 were excited at 510 nm and 610 nm, respectively. Emission was collected using a 371 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n14 \n620/60 nm bandpass filter for LD555 and a BG4 700 nm long-pass filter for LD655. Photon arrival 372 \ntimes were accumulated until the TCSPC histogram reached a peak count of 50,000 photons. 373 \nFluorescence decay curves were analyzed by fitting mono- or bi-exponential decay models using 374 \nFluoFit software (PicoQuant) and amplitude weighted average of fluorescence lifetime was 375 \ncalculated. 376 \n 377 \nNanobody production and purification 378 \nThe nanobody Nb9F10 S63C was  expressed and purified as described previously 11. Briefly, Nb9F10 S63C 379 \nwas produced in E. coli BL21(DE3) cells grown in Terrific Broth (TB; Carl Roth) supplemented with 380 \n100 μg ml-1 ampicillin at 37 ˚C. At an OD 600 of 0.6, expression was induced with 1 mM IPTG, followed 381 \nby overnight incubation at 28 ˚C. Cells were harvested by centrifugation (4,500  × g, 4 ˚C, 15 min) and 382 \nstored at –80 ˚C. For purification, cell pellets were resuspended in nanobody lysis buffer (25  mM 383 \nHEPES-NaOH pH 7.4, 300 mM NaCl, 15 mM imidazole, 0.5 mM PMSF) and disrupted by sonication. 384 \nCell debris was removed by centrifugation (18,000 × g, 4 ˚C, 35  min), and the clarified lysate was 385 \napplied to Ni-NTA agarose equilibrated in potassium phosphate (KP i) buffer (25 mM KP i pH 6.5, 386 \n100 mM KCl, and 0.5 mM tris(2-carboxyethyl) phosphine (TCEP)). Bound nanobody was washed with 387 \n10 column volumes (CV) of KP i buffer and eluted with 8 CV of elution buffer (25 mM KP i pH 6.0, 388 \n20 mM KCl, 300 mM imidazole, 0.5 mM TCEP). Eluted fractions were pooled and further purified by 389 \ncation exchange chromatography (CEX) on a HiTrap SP column (Cytiva) using a linear gradient from 390 \nlow-salt buffer (25 mM KP i pH 6.0, 20 mM KCl, 0.5 mM TCEP) to high-salt buffer (25 mM KP i pH 6.0, 391 \n500 mM KCl, 0.5 mM TCEP). The purified nanobody was concentrated and buffer-exchanged into 392 \nnanobody SEC buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl) using Zeba spin desalting columns, 393 \nfollowed by SEC (Superdex 200 Increase 10/300 GL; Cytiva). For site-specific conjugation, Nb9F10 S63C 394 \nwas incubated with a biotin-PEG 11-maleimide linker (Sigma-Aldrich) at a 1.2:1 molar ratio of protein 395 \nto linker in the presence of 0.5 mM TCEP for 2 h at 4 ˚C. Excess linker was removed by desalting on 396 \nZeba Spin Desalting Columns, followed by a final SEC step (Superdex 200 Increase 10/300 GL). 397 \n 398 \nSDS-PAGE  399 \nThe purity of TmrAB samples was assessed by SDS-PAGE. Resolving gels (12%) were prepared using 400 \n12% (w/v) acrylamide, 0.5 M Tris-HCl (pH 8.8), 0.13% (w/v) SDS, 0.05% (w/v) ammonium persulphate 401 \n(APS), and 0.25% (v/v) N,N,N’,N’-tetramethylethylenediamine (TEMED). Stacking gels contained 4.3% 402 \n(w/v) acrylamide, 0.5 M Tris/HCl (pH 6.8), 0.09% (w/v) SDS, 0.09% (w/v) APS, and 0.33% (v/v) TEMED. 403 \nGels were used immediately or stored at 4 ˚C for up to 4 weeks. Protein samples were mixed with 4× 404 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n15 \nSDS loading buffer containing dithiothreitol (DTT; Sigma-Aldrich) and heated at 90 ˚C for 5 min before 405 \nloading. Electrophoresis was performed at a constant voltage of 120 V using 1× SDS running buffer 406 \n(25 mM Tris-HCl pH 8.8, 192 mM glycine, 0.1% SDS). Proteins were visualized by staining with 407 \nInstantBlueTM Protein Stain (Abcam) for 1 h at room temperature with gentle agitation and imaged 408 \nusing a Fusion FX system (Vilber). 409 \n 410 \nATPase activity assay 411 \nThe ATPas e activity of β -DDM-solubilized TmrAB wt was quantified using a Malachite Green-based 412 \ncolorimetric assay as described previously 44. Detergent-solubilized TmrAB (0.6 μM) was incubated in 413 \nATPase buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 2 mM MgCl 2, 1 mM β-DDM) containing 414 \n3 mM ATP (Sigma-Aldrich) at 40 ˚C for 7 min. Autohydrolysis controls were prepared by incubation of 415 \nATP in ATPase buffer without protein. Reactions were quenched by adding 20 mM H 2SO4, followed 416 \nby incubation with 3 mM Malachite Green (Thermo Fisher Scientific), 0.2% (v/v) Tween20 (Carl Roth), 417 \nand 1.5% (w/v) ammonium molybdate (Carl Roth) for 10 min at room temperature. The absorbance 418 \nat 620 nm was recorded on a CLARIOstar v.5.20 R5 plate reader (BMG LABTECH). 419 \n 420 \nEnsemble FRET measurements 421 \nThe ATP binding and FRET characteristics of selected TmrAB variants were assessed by ensemble 422 \nFRET. Labeled TmrAB (100 nM) was incubated with increasing concentrations of ATP at 42 ˚C for 423 \n5 min. Donor-excited emission was recorded from 550 –700 nm with an excitation wavelength of 424 \n520 nm using a Clariostar v.5.20 R5 plate reader (BMG LABTECH). Acceptor emission intensities at 425 \n675 nm were plotted against ATP concentration and fitted with a hyperbolic function to determine 426 \nthe apparent dissociation constant (Kd, ATP) for each variant. 427 \n 428 \nFunctionalization of glass slides for single-molecule FRET analysis 429 \nGlass coverslips used for TmrAB immobilization in smFRET experiments were functionalized by 430 \nPEGylation as described previously 45. Coverslips (Carl Roth) were cleaned by sequential sonication in 431 \nMilli-Q water and analytical-grade acetone (>99.9%; VWR International), followed by oxygen plasma 432 \ntreatment (0.3 mbar, 80% power, 15 min) using a Zepto plasma cleaner (Diener) and a 10 min 433 \nincubation in methanol (Avantor, Gliwice, PL). Coverslips were then silanized by incubation for 434 \n30 min in a solution of 100 ml methanol, 5 ml acetic acid, and 3 ml 3-aminopropyltrimethoxysilane 435 \n(APTES; Tokyo Chemical Industry). After silanization, sides were rinsed four times with methanol and 436 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n16 \ndried under a nitrogen stream. Surface functionalization was achieved using a mixture of 437 \nbiotinylated-PEG (4 mol%) and nonbiotinylated-PEG (96 mol%; Rapp Polymer). The PEG solution was 438 \nsandwiched between two coverslips and incubated overnight in a humidity chamber. Coverslips were 439 \nthen rinsed thoroughly with Milli-Q water and dried under nitrogen. To enhance passivation, a 440 \nsecond PEGylation step was performed using 25 mM CH 3-PEG-NHS (333 Da; Thermo Fisher Scientific) 441 \nunder the same conditions. Finally, slides were rinsed with Milli-Q water, dried under nitrogen, and 442 \nstored at –20 ˚C under argon until use. 443 \n 444 \nSingle-molecule FRET imaging 445 \nSingle-molecule FRET (smFRET) experiments were performed using a flow chamber system (Ibidi). 446 \nChambers were assembled by placing a biotin-PEG- functionalized glass slide onto a μ -Slide I Luer 447 \nFamily flow channel (Ibidi), with the functionalized surface facing inward. All buffers and Milli-Q 448 \nwater were filtered through 0.2 μm filters (Sigma -Aldrich). Chambers were washed with 1 ml Milli-Q 449 \nwater and incubated with 0.2 mg ml-1 streptavidin (Sigma-Aldrich) at 4 ˚C for 30 min to allow binding 450 \nto the biotin-PEG surface. Unbound streptavidin was removed by washing with 1 ml Milli-Q water. 451 \nThe surface was then treated with 0.3 mg ml-1 biotinylated-PEG 11-Nb9F10S63C at 4 ˚C for 45  min, 452 \nfollowed flushing with 2 ml SEC buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM β -DDM). 453 \nDetergent-solubilized, fluorophore-labeled TmrAB (100 nM) was added and incubated at 4 ˚C for 1 h. 454 \nUnbound protein was removed by five washes with 1 ml TmrAB-SEC buffer. Chambers were 455 \nequilibrated with 1  ml of imaging buffer containing 25 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 456 \n3 mM MgCl 2, 50 mM glucose, 5 mM Trolox, 7.5 U ml-1 pyranose oxidase, and 1 kU ml-1 catalase, 457 \nsupplemented with the desired ATP concentration. For EDTA trapping experiments, MgCl 2 was 458 \nomitted and replaced with 3 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldri ch). smFRET 459 \ndata were acquired at 40 ˚C using alternating laser excitation (ALEX) on a total internal reflection 460 \nfluorescence (TIRF) microscope (NanoImager S, ONI, Oxford, UK). Typically, 600 frames were 461 \nrecorded per region of interest (ROI) with 100 ms exposure time. Laser powers were 0.8 mW cm-2 462 \n(532 nm) and 0.9 mW cm-2 (640 nm). Data were recorded in 1-min intervals, except for TmrAB NBD 463 \nvariant in apo and 3 mM ATP conditions, where 3-min intervals were used to confirm that 464 \nconformation transitions do not occur on timescales longer that one minute due to the reduced 465 \ntemperature. 466 \n 467 \nSingle-molecule FRET data analysis 468 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n17 \nSingle-molecule FRET (smFRET) measurements were performed using alternating laser excitation 469 \n(ALEX), allowing assignment of detected photons based on both excitation and emission 470 \nwavelengths. Photon counts for each molecule were extracted using NanoImager software (ONI 471 \nNanoImager, Development Build) and classified into three detection channels: donor excitation with 472 \ndonor emission (𝑓𝐷𝑒𝑥\n𝐷𝑒𝑚), donor excitation with acceptor emission ( 𝑓𝐷𝑒𝑥\n𝐴𝑒𝑚), and acceptor excitation with 473 \nacceptor emission (𝑓𝐴𝑒𝑥\n𝐴𝑒𝑚). Traces were analyzed with DeepFRET 35 and manually curated. To minimize 474 \nbias, a second researcher independently curated traces from both ATP-free and 3 mM ATP samples, 475 \nyielding 98% overlap between curations. FRET efficiency (E) and stoichiometry (S) were calculated as: 476 \nPopulation analysis was performed by constructing one-dimensional histograms of FRET efficiency (E) 477 \nand stoichiometry ( S) using OriginPro 2024 (OriginLab). Histograms were fitted with two Gaussian 478 \ndistributions corresponding to the ATP-free state (defined from apo samples) and the ATP-bound 479 \nstate (defined by a two-component fit at saturating ATP). Hidden Markov Modeling (HMM) of 480 \nindividual traces was performed using MASH-FRET 38 to distinguish dynamic from static molecules 481 \nwithin each sample. 482 \n 483 \nATP-bound dwell times and distribution of conformational states 484 \nThe ATP-bound dwell time (τd) was estimated as: 485 \nwhere fATP-bound is the fraction of ATP-bound molecules derived from Gaussian fits of the FRET 486 \nefficiency histograms ( Fig. 3). This approach assumes (i) ATP hydrolysis occurs exclusively at the 487 \ncanonical NBS, with negligible contribution from the noncanonical site 9,11,14, and (ii) the majority of 488 \nmolecules are catalytically competent and continuously cycling.  489 \nThe distribution of conformational states within ATP-bound FRET population ( E = 0.86) of TmrAB PG 490 \nunder turnover conditions (3 mM ATP) was determined using a two-state model: 491 \n𝐸 =  \n𝑓𝐷𝑒𝑥\n𝐴𝑒𝑚\n𝑓𝐷𝑒𝑥\n𝐷𝑒𝑚 + 𝑓𝐴𝑒𝑥\n𝐴𝑒𝑚\n \nEq. 1 \n𝑆 =  \n𝑓𝐷𝑒𝑥\n𝐷𝑒𝑚 + 𝑓𝐷𝑒𝑥\n𝐴𝑒𝑚\n𝑓𝐷𝑒𝑥\n𝐷𝑒𝑚 +  𝑓𝐷𝑒𝑥\n𝐴𝑒𝑚 + 𝑓𝐴𝑒𝑥\n𝐴𝑒𝑚\n \nEq. 2 \n \n𝜏𝑑 =  𝜏𝑐𝑦𝑐𝑙𝑒 × 𝑓𝐴𝑇𝑃−𝑏𝑜𝑢𝑛𝑑  Eq. 3 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n18 \nwhere fopen is the fraction of OF open state (E open = 0.63) readily resolved under trapping conditions 492 \n(Fig. 4), and fother represents the combined fraction of PG-closed states (OF occluded/URasym/URasym*, 493 \nEother = 0.97).   494 \n𝐸 =  𝑓𝑜𝑝𝑒𝑛 × 𝐸𝑜𝑝𝑒𝑛 + 𝑓𝑜𝑡ℎ𝑒𝑟 × 𝐸𝑜𝑡ℎ𝑒𝑟 Eq. 4 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n19 \nReferences 495 \n1. Davidson AL, Dassa E, Orelle C, Chen J. Structure, function, and evolution of bacterial ATP-496 \nbinding cassette systems. Microbiol Mol Biol Rev 72, 317–364 (2008). 497 \n2. Rees DC, Johnson E, Lewinson O. ABC transporters: the power to change. Nat Rev Mol Cell Biol  498 \n10, 218–227 (2009). 499 \n3. Thomas C, Tampé R. Structural and mechanistic principles of ABC transporters. Annu Rev 500 \nBiochem 89, 605–636 (2020). 501 \n4. Locher KP. Mechanistic diversity in ATP-binding cassette (ABC) transporters. Nat Struct Mol Biol  502 \n23, 487–493 (2016). 503 \n5. Thomas C , et al.  Structural and functional diversity calls for a new classification of ABC 504 \ntransporters. FEBS Lett 594, 3767–3775 (2020). 505 \n6. Robey RW, Pluchino KM, Hall MD, Fojo AT, Bates SE, Gottesman MM. Revisiting the role of ABC 506 \ntransporters in multidrug-resistant cancer. Nat Rev Cancer 18, 452–464 (2018). 507 \n7. Abele R, Tampé R. The ABCs of immunology: structure and function of TAP, the transporter 508 \nassociated with antigen processing. Physiology (Bethesda) 19, 216–224 (2004). 509 \n8. Kim J , et al. Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC 510 \nexporter. Nature 517, 396–400 (2015). 511 \n9. Zutz A , et al.  Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport 512 \ncomplex TmrAB from Thermus thermophilus. J Biol Chem 286, 7104–7115 (2011). 513 \n10. Nöll A , et al.  Crystal structure and mechanistic basis of a functional homolog of the antigen 514 \ntransporter TAP. Proc Natl Acad Sci U S A 114, E438–E447 (2017). 515 \n11. Hofmann S , et al.  Conformation space of a heterodimeric ABC exporter under turnover 516 \nconditions. Nature 571, 580–583 (2019). 517 \n12. Stefan E, Hofmann S, Tampé R. A single power stroke by ATP binding drives substrate 518 \ntranslocation in a heterodimeric ABC transporter. eLife 9, e55943 (2020). 519 \n13. Stefan E , et al. De novo macrocyclic peptides dissect energy coupling of a heterodimeric ABC 520 \ntransporter by multimode allosteric inhibition. eLife 10, e67732 (2021). 521 \n14. Barth K, Rudolph M, Diederichs T, Prisner TF, Tampé R, Joseph B. Thermodynamic basis for 522 \nconformational coupling in an ATP-binding cassette exporter. J Phys Chem Lett  11, 7946 –7953 523 \n(2020). 524 \n15. Barth K, Hank S, Spindler PE, Prisner TF, Tampé R, Joseph B. Conformational coupling and trans-525 \ninhibition in the human antigen transporter ortholog TmrAB resolved with dipolar EPR 526 \nspectroscopy. J Am Chem Soc 140, 4527–4533 (2018). 527 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n20 \n16. Agam G, et al. Reliability and accuracy of single-molecule FRET studies for characterization of 528 \nstructural dynamics and distances in proteins. Nat Methods 20, 523–535 (2023). 529 \n17. Hellenkamp B , et al.  Precision and accuracy of single-molecule FRET measurements-a multi-530 \nlaboratory benchmark study. Nat Methods 15, 669–676 (2018). 531 \n18. Sasmal DK, Pulido LE, Kasal S, Huang J. Single-molecule fluorescence resonance energy transfer 532 \nin molecular biology. Nanoscale 8, 19928–19944 (2016). 533 \n19. Bartels K, Lasitza-Male T, Hofmann H, Löw C. Single-molecule FRET of membrane transport 534 \nproteins. ChemBioChem 22, 2657–2671 (2021). 535 \n20. Lerner E , et al. FRET-based dynamic structural biology: Challenges, perspectives and an appeal 536 \nfor open-science practices. eLife 10, e60416 (2021). 537 \n21. Nettels D, Galvanetto N, Ivanović MT, Nüesch M, Yang T, Schuler B. Single -molecule FRET for 538 \nprobing nanoscale biomolecular dynamics. Nat Rev Phys 6, 587–605 (2024). 539 \n22. Wang L, Johnson ZL, Wasserman MR, Levring J, Chen J, Liu S. Characterization of the kinetic cycle 540 \nof an ABC transporter by single-molecule and cryo-EM analyses. eLife 9, e56451 (2020). 541 \n23. Levring J, Terry DS, Kilic Z, Fitzgerald G, Blanchard SC, Chen J. CFTR function, pathology and 542 \npharmacology at single-molecule resolution. Nature 616, 606–614 (2023). 543 \n24. Husada F , et al. Conformational dynamics of the ABC transporter McjD seen by single-molecule 544 \nFRET. EMBO J 37, e100056 (2018). 545 \n25. Nocker C, Pečak M, Nocker T, Fahim A, Sušac L, Tampé R. Single -molecule dynamics reveal ATP 546 \nbinding alone powers substrate translocation by an ABC transporter. BioRxiv, 547 \n2025.2011.2027.690960 (2025). 548 \n26. Grossmann N, Vakkasoglu AS, Hulpke S, Abele R, Gaudet R, Tampé R. Mechanistic determinants 549 \nof the directionality and energetics of active export by a heterodimeric ABC transporter. Nat 550 \nCommun 5, 5419 (2014). 551 \n27. Sun P , et al.  Substrate recognition diversity and transport dynamics of ABCC1. Nat Commun 16, 552 \n10499 (2025). 553 \n28. Altman RB , et al. Cyanine fluorophore derivatives with enhanced photostability. Nat Methods 9, 554 \n68–71 (2011). 555 \n29. Martin MI , et al. Leveraging Baird aromaticity for advancement of bioimaging applications. J Phys 556 \nOrg Chem 36,  (2023). 557 \n30. Kalinin S , et al.  A toolkit and benchmark study for FRET-restrained high-precision structural 558 \nmodeling. Nat Methods 9, 1218–1225 (2012). 559 \n31. Ha T, Tinnefeld P. Photophysics of fluorescent probes for single-molecule biophysics and super-560 \nresolution imaging. Annu Rev Phys Chem 63, 595–617 (2012). 561 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n21 \n32. Sindbert S , et al.  Accurate distance determination of nucleic acids via Förster resonance energy 562 \ntransfer: implications of dye linker length and rigidity. J Am Chem Soc 133, 2463–2480 (2011). 563 \n33. Dale RE, Eisinger J, Blumberg WE. The orientational freedom of molecular probes. The 564 \norientation factor in intramolecular energy transfer. Biophys J 26, 161–193 (1979). 565 \n34. van der Meer BW. Kappa-squared: from nuisance to new sense. J Biotechnol 82, 181–196 (2002). 566 \n35. Thomsen J , et al. DeepFRET, a software for rapid and automated single-molecule FRET data 567 \nclassification using deep learning. eLIfe 9, e60404 (2020). 568 \n36. Hohlbein J, Craggs TD, Cordes T. Alternating-laser excitation: single-molecule FRET and beyond. 569 \nChem Soc Rev 43, 1156–1171 (2014). 570 \n37. Asher WB , et al.  Single-molecule FRET imaging of GPCR dimers in living cells. Nat Methods 18, 571 \n397–405 (2021). 572 \n38. Hadzic M, Borner R, Konig SLB, Kowerko D, Sigel RKO. Reliable state identification and state 573 \ntransition detection in fluorescence intensity -based single -molecule Förster Resonance Energy -574 \nTransfer data. J Phys Chem B 122, 6134–6147 (2018). 575 \n39. Dimura M, Peulen TO, Hanke CA, Prakash A, Gohlke H, Seidel CA. Quantitative FRET studies and 576 \nintegrative modeling unravel the structure and dynamics of biomolecular systems. Curr Opin 577 \nStruct Biol 40, 163–185 (2016). 578 \n40. Lam K, Tajkhorshid E. Membrane interactions of Cy3 and Cy5 fluorophores and their effects on 579 \nmembrane-protein dynamics. Biophys J 119, 24–34 (2020). 580 \n41. Grabenhorst L, Sturzenegger F, Hasler M, Schuler B, Tinnefeld P. Single-molecule FRET at 10 MHz 581 \ncount rates. J Am Chem Soc 146, 3539–3544 (2024). 582 \n42. Bonhomme L , et al. Triple labeling resolves a GPCR intermediate state by using three-color single 583 \nmolecule FRET. J Am Chem Soc 147, 17689–17700 (2025). 584 \n43. Yim SW , et al.  Four-color alternating-laser excitation single-molecule fluorescence spectroscopy 585 \nfor next-generation biodetection assays. Clin Chem 58, 707–716 (2012). 586 \n44. Diederichs T, Tampé R. Single cell-like systems reveal active unidirectional and light-controlled 587 \ntransport by nanomachineries. ACS Nano 15, 6747–6755 (2021). 588 \n45. Chandradoss SD, Haagsma AC, Lee YK, Hwang JH, Nam JM, Joo C. Surface passivation for single-589 \nmolecule protein studies. J Vis Exp, 50549 (2014). 590 \n46. Chaptal V , et al.  Substrate-bound and substrate-free outward-facing structures of a multidrug 591 \nABC exporter. Sci Adv 8, eabg9215 (2022). 592 \n  593 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n22 \nAuthor contributions  594 \nM.P. prepared all TmrAB samples and carried out the experiments for this study. M.P. performed 595 \ndata analysis. Curated traces were independently checked by C.N. to avoid human bias. M.P. and 596 \nC.N. prepared functionalized glass slides for single-molecule FRET . M.P. and R.T. wrote the 597 \nmanuscript. R.T. conceived and supervised the work.  598 \n 599 \nAcknowledgements 600 \nThis work was supported by the European Research Council (ERC Advanced Grant 101141396 to 601 \nR.T.), the German Research Foundation via the Collaborative Research Center CRC 1507/P18 to R.T. 602 \nand the Research Training Group (GRK 1986/B4.7 to R.T.). We thank Jan F.M. Stuke and Jonas 603 \nGöhmann for their support in automating trace extraction from ONI NanoImager software, Dr. David 604 \nGlück for guidance on lifetime measurements, and Tobias Nocker for preparing nanobodies used in 605 \nthe study. We are also grateful to the Wachtveitl lab (Goethe University Frankfurt) for access to their 606 \nFluoTime 100 spectrometer (PicoQuant). Finally, we thank Dr. Rupert Abele, Dr. David Glück, Dr. 607 \nSimon Trowitzsch, Inga Nold, and Andrea Pott for helpful comments on the manuscript and 608 \nproofreading. 609 \n 610 \nData and materials availability 611 \nAll data are available in the main text or the supplementary materials. All other data are available 612 \nfrom the corresponding author upon reasonable request. Source data are provided with this paper: 613 \nDOI: 614 \nhttps://doi.org/10.25716/gude.0jbq-k1q6. 615 \n 616 \nCompeting Interest 617 \nThe authors declare no competing interest.  618 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n23 \nFigures 619 \n 620 \nFigure 1. Accessible-volume (AV) simulations of LD fluorophores on TmrAB variants.  AV simulations 621 \nwere performed for LD555 (donor) and LD655 (acceptor) fluorophores attached to the selected 622 \nTmrAB labeling sites to assess whether donor-acceptor distances are suitable for smFRET 623 \nmeasurements30. a, TmrAB NBD (TmrA C416BL458C) and b, TmrABPG (TmrA C416A, T61C BR56C) in the inward-624 \nfacing wide ( IFwide; PDB: 6RAN, left) and outward-facing open ( OFopen; PDB: 6RAH, right) 625 \nconformations. Approximate membrane position is indicated by the dashed grey line. For all 626 \nsimulations, TmrA is shown in blue with LD655 (orange) and TmrB in yellow with LD555 (green). c, AV 627 \nsimulations confirmed that donor-acceptor distances ( RDA) remain within the FRET-sensitive range in 628 \nboth conformations, predicting measurable shifts in simulated FRET efficienc ies ( Esim). Cryo-EM 629 \nstructures of TmrAB reconstituted in lipid nanodiscs 11 were used as templates . Structures were 630 \ndetermined either in apo state (apo) or in presence of 3 mM ATP (turnover) . Outward-facing open 631 \n(OFopen) and outward-facing occluded ( OFoccluded) structures were obtained via orthovanadate 632 \ntrapping (Vi) or by using the slow-turnover catalytic mutant TmrAE523QB (EQ).  633 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n24 \n 634 \nFigure 2. ATP-induced conformational changes of TmrAB analyzed by smFRET. a, Experimental 635 \nsetup. TmrABNBD (left) and TmrAB PG (right) variants were labeled with LD555/LD655 and tethered to 636 \nPEGylated coverslips via a biotinylated anti-TmrB nanobody ( Nb9F10S63C). b, smFRET imaging. 637 \nSamples were recorded using total internal reflection fluorescence (TIRF) microscopy with alternating 638 \nlaser excitation (ALEX ; donor: 532 nm; acceptor: 640 nm). Emission was collected in two channels 639 \n(donor: 498-620 nm; acceptor: 662-710 nm) . Donor fluorescence ( 𝑓𝐷𝑒𝑥\n𝐷𝑒𝑚: donor excitation at donor 640 \nemission), sensitized acceptor fluorescence ( 𝑓𝐷𝑒𝑥\n𝐴𝑒𝑚: donor excitation at acceptor emission), and direct 641 \nacceptor fluorescence ( 𝑓𝐴𝑒𝑥\n𝐴𝑒𝑚: acceptor excitation at acceptor emission) were used to calculate FRET 642 \nefficiency ( E) and stoichiometry ( S). Fluorescence time traces were analyzed with DeepFRET 35. 643 \nc, Representative TmrAB NBD trace in ATP-bound state (top) and corresponding FRET 644 \nefficiency/stoichiometry plot (bottom). Donor emission upon donor excitation is shown in green as 645 \nthe number of detected photons per frame (counts), acceptor intensity upon donor excitation in 646 \norange, FRET efficiency ( E) in black, and stoichiometry ( S) in grey. The slight increase in donor 647 \nfluorescence observed during the first seconds of acquisition reflects photophysical equilibration of 648 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n25 \nthe fluorophore and instrumental stabilization and does not affect FRET efficiency or stoichiometry, 649 \nwhich are ratio-based and remain constant over time 17. d,e, Population analysis. FRET efficiency (E ) 650 \nhistograms for (d) TmrABNBD and (e) TmrABPG are shown for the apo state (top) and ATP-bound state 651 \n(bottom; 3 mM ATP ). Histograms were fitted with two Gaussian populations corresponding to the 652 \napo state (blue; defined from apo measurements) and the ATP-bound state (orange; defined from 653 \ntwo-component fits at saturati ng ATP). Dotted vertical lines indicate mean E values of each 654 \npopulation. Population fractions , calculated from Gaussian areas, are summarized schematically in 655 \neach panel.  656 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n26 \n 657 \nFigure 3. ATP-dependent shifts in smFRET populations of TmrAB.  a,c, Increasing ATP concentrations 658 \ngradually redistributed the population between apo and ATP-bound conformations for ( a) TmrABNBD 659 \nand ( c) TmrAB PG. FRET efficiency ( E) histograms were fitted with two Gaussian populations 660 \ncorresponding to the ATP-free state ( blue: defined from apo samples) and the ATP-bound state 661 \n(orange; determined from fits at saturating ATP). Dotted vertical lines indicate the mean E values of 662 \neach population. Relative proportion fractions, calculated from Gaussian areas, are summarized 663 \nschematically in each panel. b,d, ATP-binding curves were obtained by plotting the fraction of 664 \nmolecules in the ATP-bound states as a function of ATP concentration for ( b) TmrAB NBD (reporting 665 \nNBD dimerization) and ( d) TmrAB PG (reporting PG opening). Data were fitted with a Langmuir 666 \nisotherm to determine the apparent dissociation constant Kd, ATP of each variant.  667 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n27 \n 668 \nFigure 4. Iden tification of the outward-facing open ( OFopen) conformation.  a–c, Three 669 \ncomplementary approaches were employed to resolve OFopen state: ( a) the slow-turnover variant 670 \nTmrABPG_EQ, (b) imaging in Mg2+-free buffer supplemented with EDTA, and ( c) stabilizing via reverse 671 \ninhibition using high concentrations of peptide substrate. FRET efficiency ( E) histograms were fitted 672 \nwith three Gaussian populations corresponding to the ATP-free state (blue) , the ATP-bound state 673 \n(orange), and the stabilized OF open state (green) . All three strategies revealed a distinct OFopen 674 \npopulation. Dotted vertical lines indicate the mean E values of each population. Relative proportion 675 \nfractions, calculated from Gaussian areas, are summarized schematically in each panel. d, 676 \nComparison of inter-residues distances. Distances (Å) between selected residues on the NBDs and PG 677 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n28 \nof TmrAB were determined by multiple methods: smFRET values (this study) for detergent-678 \nsolubilized TmrAB; c ryo-EM distances (C β–Cβ) from nanodisc-reconstituted TmrAB (PDB 6RAH, 679 \n6RAN)11; accessible-volume (AV) simulation distances for nanodisc-reconstituted TmrAB (this study) ; 680 \nand PELDOR/DEER distances from detergent-solubilized TmrAB15.  681 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n29 \n 682 \nFigure 5. Conformational state distribution and catalytic cycle of TmrAB under active turnover.  683 \nSchematic of the TmrAB transport cycle summarizing major conformational states and their 684 \nestimated population distributions under physiological ATP concentrations (3 mM, 40 ˚C). a, The 685 \ninward-facing apo state (IF narrow and IF wide; blue arc) accounts for ~20% of molecules and is 686 \ncharacterized by separated NBDs and a cytosol-accessible substrate-binding cavity. Substrate binding 687 \nstabilizes the IFwide conformation11. Independent of substrate, ATP binding induces NBD dimerization 688 \nand transition to the ATP-bound ensemble. b,c, Under substrate-bound turnover conditions, TmrAB 689 \nproceeds via the ( b) OFoccluded to (c) OFopen state in which the substrate release occurs . Under steady-690 \nstate turnover, the ATP-bound ensemble rapidly interconverts between substrate-free OFoccluded and 691 \nOFopen accounting for ~25% of the ATP-bound population (green circle). These transitions occur faster 692 \nthan the ~200 ms temporal resolution of standard smFRET measurement, resulting in an averaged 693 \nsignal under turnover conditions. OF occluded likely serves as an obligate intermediate between IF and 694 \nOFopen, preventing substrate backflow by maintaining a substrate-binding cavity occluded during the 695 \nstructural rearrangements of the PG and NBDs. Although a substrate-bound OF occluded state has not 696 \nbeen directly observed for TmrAB, its existence is supported by structures of the homodimeric 697 \ntype IV transporter BmrA 46. Reduced ATP hydrolysis or substrate trans-inhibition enables trapping of 698 \nthe transporter in the OFopen. state. d,e, ATP hydrolysis and phosphate (P i) release generate post-699 \nhydrolysis return states: ( d) URasym and ( e) URasym*. Subsequent ADP release restores the apo IF 700 \nconformation, completing the transport cycle. Overall, the ATP-bound phase ( b–e) represents ~55% 701 \noccupancy (orange arc) with an estimated dwell time of ~310 ms, whereas the apo/ATP-rebinding 702 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n30 \nphase (a) lasts ~90 ms, yielding a total cycle time of ~400 ms ( kcat = 2.57 s⁻¹). TmrA is shown in blue, 703 \nTmrB in yellow, substrate as a green diamond, and nucleotides as orange symbols. Dotted grey boxes 704 \nindicate the approximate position of the NBD dimer interface.   705 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n31 \nFigure supplements 706 \n 707 \nFigure 1–Figure supplement 1. Quality of TmrAB purification and fluorophore labeling.  a, SDS-PAGE 708 \nanalysis (10%, reducing conditions, Coomassie staining) of successive purification steps: M, molecular 709 \nweight marker; L, cell lysate; B, Ni-NTA beads after incubation with lysate; FT, flow-through; W, 710 \nwash; E, eluted TmrAB ; BE, TmrAB after buffer exchange; C, concentrated TmrAB; CFT, concentrator 711 \nflow-through; 1 and 2, first and second peaks eluted from size-exclusion chromatography (SEC). Only 712 \nthe second peak was used for subsequent FRET experiments . b, SEC (Superdex 200 increase 10/300 713 \nGL) confirming monodispersity of labeled TmrAB and efficient removal of free fluorophores . 714 \nRepresentative chromatogram is shown for TmrAB PG. c, ATP hydrolysis activity of purified wild-type 715 \nTmrAB ( 60 nM TmrAB wt) measured at 40 ˚C for 7 min. Released inorganic phosphate (P i) was 716 \nquantified using the Malachite Green assay. Data were fitted to a Michaelis-Menten model, yielding 717 \nKm = 0.97 ± 0.28 mM and kcat = 2.57 ± 0.38 s-1. d–f, Analytical SEC (Superdex 200 increase 3.2/300) 718 \nused to determine fluorophore labeling efficienc ies for each variant: ( d) TmrABNBD, LD555: ~ 55%, 719 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n32 \nLD655: ~ 53%; ( e) TmrAB PG, LD555: ~ 43%, LD655: ~52%; and (f ) TmrAB PG_EQ, LD555: ~ 42%, LD655: 720 \n~52%.  721 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n33 \n 722 \nFigure 1–Figure supplement  2. FRET capabilities of labeled TmrAB variants.  a, Time-correlated 723 \nsingle-photon counting histograms of LD555 (left) and LD655 (middle ) measured under three 724 \nconditions: free dye in buffer (black), LD555/LD655-labeled TmrAB NBD (orange), and LD555/LD655-725 \nlabeled TmrAB PG (blue). Amplitude-weighted average fluorescence lifetimes are summarized in the 726 \ntable (right), confirming sufficient rotational freedom for reliable FRET measurements . b–d, 727 \nEnsemble donor-exited emission spectra (550 –700 nm, excitation 520 nm) of ( b) TmrAB NBD, ( c) 728 \nTmrABPG, and (d) the slow-turnover variant TmrAB PG_EQ, stochastically labeled with LD555/LD655 and 729 \nincubated with increasing ATP concentrations. Spectra are normalized to donor intensity in the apo 730 \nstate. ATP-dependent donor quenching and acceptor sensitization indicate that all variants retain 731 \nFRET capability. e–g, Fractional fluorescence change s, ( F-F₀)/F₀, where F is acceptor emission 732 \nintensity and F₀ is the intensity in the apo state, plotted as a function of ATP concentration for ( e) 733 \nTmrABNBD, ( f) TmrAB PG, and ( g) TmrAB PG_EQ. Data were fitted with a hyperbolic binding model to 734 \ndetermine apparent Kd, ATP values, consistent with ensemble FRET measurements of ATP binding.  735 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n34 \n 736 \nFigure 2–Figure supplement 1. Representative  smFRET traces of TmrAB NBD. Representative single-737 \nmolecule FRET (smFRET) traces of TmrAB NBD were recorded ( a) in the ATP-free state and ( b, c) in the 738 \npresence of 3 mM ATP. Hidden Markow modeling (HMM) was applied to classify traces into (b ) static 739 \nand (c) dynamic, based on the absence or presence of transitions between ATP-free and ATP-bound 740 \nconformational states. Donor fluorescence intensity upon donor excitation is shown in green, 741 \nacceptor fluorescence intensity upon donor excitation in orange , FRET efficiency ( E) in black, and 742 \nstoichiometry (S) in grey.  743 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint \n\n35 \n 744 \nFigure 2–Figure supplement 2. Representative  smFRET traces of TmrAB PG. Representative single-745 \nmolecule FRET (smFRET) traces of TmrAB PG were recorded (a ) in the ATP-free state and ( b, c) in the 746 \npresence of 3 mM ATP. Hidden Markow modeling (HMM) was applied to classify traces into ( b) static 747 \nand (c) dynamic, based on the absence or presence of transitions between ATP-free and ATP-bound 748 \nconformational states. Donor fluorescence intensity upon donor excitation is shown in green, 749 \nacceptor fluorescence intensity upon donor excitation in orange, FRET efficiency ( E) in black, and 750 \nstoichiometry (S) in grey. 751 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}