ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter

7 ATP-binding cassette (ABC) transporters are essential molecular machines whose conformational 8 dynamics have largely been inferred from ensemble-averaged measurements . Resolving dynamic 9 heterogeneity and transient intermediates, however, requires single-molecule approaches. Here, we 10 use single-molecule Förster resonance energy transfer (smFRET) to directly monitor conformational 11 changes of the heterodimeric type IV ABC transporter TmrAB, a functional homolog of the human 12 antigen transporter TAP . Fluorophores positioned at the nucleotide-binding domains and the 13 periplasmic gate were validated by accessible-volume simulations, fluorescen ce lifetimes, and 14 ensemble FRET, demonstrating that these reporters reliably track conformational transitions . Single-15 molecule analysis distinguishes ATP-free and ATP-bound states and reveals ATP-dependent 16 population shifts from nucleotide-free to physiological ATP concentrations . Probing conformational 17 dwell-times further uncovers an unexpectedly long ATP-bound dwell time of ~300 ms. Using 18 complementary stabilization strategies –including a slow-turnover variant, Mg²⁺ depletion, o r 19 substrate trans-inhibition –we resolve a previously hidden outward-facing open state that rapidly 20 interconverts with occluded intermediates under turnover conditions. These results provide the first 21 single-molecule characterization of TmrAB and establish a general framework for dissecting ATP-22 coupled conformational dynamics in heterodimeric ABC transporters. 23 24

ABC transporters, conformational dynamics, membrane protein, single-molecule analysis 25 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 2

26 ATP-binding cassette (ABC) transporters constitute the largest family of primary active membrane 27 transport systems, conserved across all domains of life1-3. Despite considerable structural diversity, all 28 ABC transporters share a modular architecture compris ing two conserved nucleotide-binding 29 domains (NBDs)–the defining hallmark of the family –and two transmembrane domains (TMDs) that 30 form the substrate translocation pathway 3,4. Based on their transmembrane-domain architecture , 31 ABC transporters are classified into seven types that encompass importers, exporters, extractors, and 32 mechanotransmitters5. Substrate translocation is driven by large conformational changes that are 33 chemo-mechanically coupled to ATP binding, hydrolysis, and phosphate/ADP release 2,3. ABC 34 transporters play central roles in cellular homeostasis, nutrient uptake, waste removal, and toxin 35 defense. Their dysfunction and misregulation are linked to numerous diseases and drug resistance6. 36 The heterodimeric type IV ABC transporter TmrAB from Thermus thermophilus has emerged as a 37 powerful model system due to its exceptional thermal stability and functional homology to the 38 transporter associated with antigen processing (TAP1/2), a key component of adaptive immunity 7-9. 39 Notably, TmrAB shares overlapping peptide specificity with TAP and can restore antigen presentation 40 in TAP-deficient human cells 10. Its inherent asymmetry, with one catalytically active (canonical) and 41 one inactive (noncanonical) nucleotide-binding site (NBS), provides a unique opportunity to 42 investigate functional specialization and asymmetry in ABC transport mechanisms. 43 Extensive structural studies, particularly using cryogenic electron microscopy (cryo-EM), ha ve 44 delineated the conformation al landscape of TmrAB and yielded a detailed model of its translocation 45 cycle11,12. In this model, TmrAB fluctuates between inward-facing wide and narrow conformations 46 (IFwide and IFnarrow), characterized by a sealed periplasmic gate (PG) and well-separated NBDs, thereby 47 permitting substrate access to the central binding cavity. ATP binding to both NBDs induces NBD 48 dimerization and drives the transition into the outward-facing states, including an OF open (OF open) 49 conformation with an open PG that enables substrate release into the periplasm, as well as an OF 50 occluded (OF occluded) state characterized by a sealed PG and dimerized NBDs. Subsequent ATP 51 hydrolysis and phosphate release lead to asymmetric unlocked return states (UR asym and URasym*), 52 before the transporter returns to the IF conformation. These UR sates feature as sealed PG, a 53 partially open ADP-bound canonical NBS, and a tightly ATP-occluded noncanonical NBS11. 54 Single-turnover experiments established that ATP binding, rather than hydrolysis, drives the IF-to-OF 55 transition, while phosphate release precedes the OF-to-IF switch 12,13. Complementary ensemble 56 approaches, including pulsed electron –electron double resonance (PELDOR/DEER) spectroscopy, 57 have further characterized ATP-dependent conformational changes 14,15. Howe ver, ensemble 58 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 3 averaging inherently masks molecular heterogeneity, obscures inactive or misfolded subpopulations, 59 and limits access to kinetic information. 60 Single-molecule techniques overcome these limitations by resolving conformational dynamics at the 61 level of individual molecules 16,17. In particular, single-molecule Förster resonance energy transfer 62 (smFRET) enables real-time monitoring of protein conformational changes with nanometer 63 precision18-21. Applied to ABC transporters, smFRET provides a unique opportunity to dissect 64 transport cycles, resolve transient intermediates, and extract kinetic and mechanistic insights that 65 remain inaccessible to ensemble-based measurement approaches22-24. 66 Here, we apply total internal reflection fluorescence (TIRF) microscopy combined with alternating 67 laser excitation (ALEX)-based smFRET to detergent-solubilized heterodimeric ABC transporter TmrAB, 68 providing the first single-molecule characterization of this system. By strategically positioning 69 fluorophore pairs, we directly monitor ATP-dependent NBD dimerization and periplasmic gate ( PG) 70 opening, quantify conformational state occupancies across ATP concentrations ranging from 71 nucleotide-free to physiological levels (3 mM), and uncover conformational dynamics previously 72 masked by ensemble averaging. Using three orthogonal trapping strategies –(i) a slow-turnover 73 catalytic mutant 11,12, (ii) Mg²⁺ depletion 14,25, and (iii) substrate trans-inhibition 26,27–we resolved a 74 previously hidden outward-facing open (OF open) state that rapidly exchanges with the outward-facing 75 occluded (OFoccluded) state. Distance measurements derived from smFRET closely match ed predictions 76 from accessible-volume (AV) simulations, cryo-EM structures, and PELDOR/DEER spectroscopy, 77 confirming that detergent-solubilized TmrAB retains a native-like conformational landscape. 78 Together, these results provide the first single-molecule quantification of conformational state 79 occupancies for a heterodimeric type IV ABC transporter and establish TmrAB as a versatile model 80 for single-molecule studies of ABC transport systems. 81 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 4

82 Design of FRET-labeled TmrAB variants to probe conformational dynamics 83 To monitor conformational changes in distinct regions of TmrAB, w e engineered FRET variants 84 targeting the nucleotide-binding domains (NBDs) and the periplasmic gate (PG). The NBDs undergo 85 ATP-dependent dimerization followed by post-hydrolysis dissociation , whereas the PG opening and 86 closing controls substrate release into the periplasm 2,3,11. Probing both regions provides 87 complementary readouts of cytosolic and periplasmic coupling during the transport cycle. 88 Labeling positions were selected based on prior PELDOR/DEER studies 15. The NBD report er variant 89 (TmrAC416BL458C, referred to as TmrAB NBD) monitors conformational changes at the noncanonical 90 nucleotide-binding site (NBS) , while the PG reporter (TmrA C416A, T61CBR56C, hereafter TmrAB PG) tracks 91 PG opening. In TmrAB NBD, the native single cysteine (C416) was retained for labeling, whereas in 92 TmrABPG it was substituted by alanine to prevent off-target labeling. Selecting the noncanonical 93 rather than the canonical NBS prevents distinguish ing outward-facing occluded (OF occluded) from 94 asymmetric unlocked return states (URasym and URasym*)11, but reduces the number of resolvable FRET 95 states and thereby simplifies data interpretation. 96 Both variants were labeled with photostable fluorophores, LD555 (donor) and LD655 (acceptor), 97 containing a 1,3,5,7-cyclooctatetraene moiety to suppress photobleaching and blinking 28,29. 98 Accessible-volume (AV) simulations 30 performed on nine cryo-EM structures11 confirmed that donor-99 acceptor distances ( RDA) and simulated FRET efficiencies ( Esim) fall within the FRET-sensitive range 100 (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 (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 0.69 ± 0.03 (53.8 ± 2.0 Å, PG open). These transitions correspond to ΔEsim values of 0.22 and 0.27 and 103 ΔRDA of 12.7 Å and 23.2 Å, for TmrAB NBD and TmrAB PG, respectively, predicting robust and 104 experimentally resolvable FRET changes. 105 Additionally, we employed a slow-turnover TmrAB variant that reports on PG opening (TmrA C416A, 106 E523Q, T61C BR56C, hereafter TmrAB PG_EQ). Substituting the catalytic glutamate with glutamine removed 107 the carboxylate required to activate water for nucleophilic attack on ATP, thus drastically reducing 108 the rate of ATP hydrolysis. This mutation slows down the catalytic turnover (~1000-fold) to a half-life 109 of approximately 25 min at 45 ˚C11,12,14,25, enabling stabilization of ATP-bound outward-facing 110 conformations. 111 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 5 TmrAB variants are suitable for FRET studies 112 TmrAB variants were expressed in E. coli and purified using immobilized metal-affinity 113 chromatography. SDS-PAGE and size-exclusion chromatography (SEC) confirmed high sample purity 114 and monodispersity (Fig. 1–Fig. S1a,b). ATPase assays of TmrABwt verified that enzymatic activity was 115 fully retained after purification, yielding a Michaelis-Menten constant ( Km) of 0.97 ± 0.28 mM and 116 catalytic ATP turnover rate (kcat) of 2.57 ± 0.38 s-1 at 40 ˚C (Fig. 1–Fig. S1c). 117 Cysteine-maleimide labeling of detergent-solubilized TmrAB variants achieved site-specific labeling 118 efficiencies exceeding 90% ( Fig. 1–Fig. S1d–f). Fluorescence lifetime ( τ) analysis of conjugated 119 fluorophores confirmed that their photophysical properties were preserved and that they retained 120 sufficient rotational freedom for reliable FRET measurements. τ histograms of both conjugated and 121 free fluorophores were fitted with a biexponential decay model, from which amplitude-weighted 122 average lifetimes were calculated. For TmrAB NBD, average τ values were 0.93 ± 0.02 ns (LD555) and 123 1.52 ± 0.01 ns (LD655), while TmrAB PG exhibited average τ values of 0.95 ± 0.02 ns (LD555) and 124 1.65 ± 0.01 ns (LD655) ( Fig. 1–Fig. S2a). By comparison, free dyes in buffer displayed lifetimes of 125 1.11 ± 0.02 ns (LD555) and 1.29 ±0.01 ns (LD655). Because the fluorescence lifetimes of both, the 126 conjugated dyes and the free dyes, remain on the ~1 ns timescale, we conclude that the 127 fluorophores remain photophysically active and are not affected by protein-induced quenching 17,31. 128 Moreover, the measured lifetimes on the nanosecond timescale are only marginally affected and, 129 most importantly, identical between the TmrAB variants, indicating dynamics orientational averaging 130 of the transition dipoles and confirming that the labeled constructs are suitable for quantitative FRET 131 studies32-34. 132 Ensemble ATP titration (0–10 mM ATP) revealed the expected concentration-dependent donor 133 quenching and acceptor sensitization ( Fig. 1–Fig. S2b–d). ATP-induced fractional fluorescence 134 changes provided as a quantitative readout of conformational transitions, allowing estimation of 135 equilibrium dissociation constants for ATP binding (Kd, ATP) to labeled TmrAB variants ( Fig. 1–Fig. S2e–136 g). The measured apparent Kd, ATP values––51 ± 38 μM for TmrAB NBD, 68 ± 25 μM for TmrAB PG, and 137 95 ± 26 μM for the slow -turnover variant TmrAB PG_EQ––are in good agreement with previously 138 reported values (~100 µM for TmrA E523QB)12, indicating that fluorophore labeling does not perturb 139 ATP binding . Notably, TmrAB PG_EQ exhibited a larger shift in ATP-induced fluorescence change than 140 TmrABPG, consistent with stabilization of the ATP-bound conformation and reduced catalytic 141 turnover. 142 143 144 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 6 145 ATP-induced conformational switching resolved by single-molecule FRET 146 TmrAB variants were site-specifically immobilized on PEGylated coverslips using a conformation-147 independent, TmrB-specific nanobody ( Nb9F10S63C)11 conjugated to maleimide-PEG 11-biotin (Fig. 2a). 148 Previous studies confirmed that this nanobody does not perturb TmrAB transport or ATPase 149 activity11,25. Donor and acceptor photons were recorded by a total-internal reflection fluorescence 150 (TIRF) microscope using alternating laser excitation (ALEX; NanoImager) at 40 ˚C ( Fig. 2b). Single-151 molecule localization , fluorescence-trajectory extraction, and background correction were 152 performed using NanoImager software, followed by DeepFRET-based machine-learning trace 153 classification and corrections for donor leakage, direct acceptor excitation, and difference in 154 detection-efficiency17,35,36 ( Fig. 2–Fig. S1 and 2). FRET efficiency ( E) and stoichiometry ( S) were 155 calculated from the corrected fluorescence-trajectories (see Methods, Eq. 1 and Eq. 2). 156 FRET efficiency (E) histograms revealed two Gaussian populations corresponding to the apo and ATP-157 bound states ( Fig. 2d,e). In the absence of ATP, only the apo population was observed, whereas 158 addition of 3 mM ATP induced the appearance of a second ATP-bound population. For TmrAB NBD, the 159 apo and ATP-bound populations exhibited mean E values of 0.58 and 0.88 ( ΔE = 0.30), respectively, 160 with ~77% of molecules occupying the ATP-bound state. For TmrAB PG, mean E values were 0.97 (apo) 161 and 0.86 (ATP-bound) (ΔE = 0.11), with ~80% of molecules in the ATP-bound state. 162 Distance estimates calculated using a Förster radius of R0 = 63.5 Å (ref.37) yielded apparent distances 163 of 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 TmrABPG. For TmrAB NBD, the experimentally derived ΔR of 14.6 Å closely agreed with the AV 165 simulations. In contrast , the smaller Δ R of 11.4 Å observed for TmrAB PG deviated from simulated 166 values, indicating that the ATP-bound population at this site represents a mixture of rapidly 167 interconverting conformations rather than a single well-defined state. 168 To assess the ATP sensitivity, we quantified conformational responses across a wide range of ATP 169 concentrations, spanning well below the reported Kd, ATP (~ 100 µM for TmrA E523QB)12 up to 170 physiologically relevant levels (3 mM ATP) . smFRET measurements revealed dose-dependent 171 population shifts: TmrAB NBD transitioned from a low-FRET apo state ( E = 0.58) to high-FRET ATP-172 bound state ( E = 0.88), whereas TmrAB PG shifted from a high-FRET apo state ( E = 0.97) to a lower-173 FRET ATP-bound state ( E = 0.86) (Fig. 3a,c). Langmuir isotherm fits yielded Kd, ATP values of 13 ± 1 μM 174 for TmrAB NBD and 2 ± 1 μM for TmrAB PG (Fig. 3b,d), indicating saturation at ATP concentrations well 175 below physiological levels (3 mM). For the TmrAB PG variant, population quantification and 176 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 7 subsequent determination of Kd, ATP are unreliable due to insufficient separation of the two FRET 177 populations below 1 mM ATP, consistent with the smaller ΔE observed for this labeling configuration. 178 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 8 Trapping of TmrABPG reveals a previously hidden outward-facing open state 179 At physiological ATP concentrations (3 mM), ~80% of TmrAB PG molecules populated the ATP-bound 180 state (E = 0.86), closely matching the ATP-bound population (~77% of NBD-dimerized) observed for 181 TmrABNBD ( Fig. 3). This agreement indicates that both labeling strategies consistently report ATP-182 dependent conformational changes. However, the ATP-induced shift observed for the periplasmic-183 gate reporter TmrABPG (ΔE = 0.11, ΔR = 11.4 Å; Fig. 3c) was substantially smaller than predicted by AV 184 simulations ( ΔE = 0.27, ΔR = 23.2 Å; Fig. 1c). This discrepancy indicates that the ATP-bound 185 population at E = 0.86 represents an unresolved ensemble, potentially comprising OFopen and 186 OFoccluded conformations, as well as the post-hydrolysis asymmetric unlocked return states (UR asym and 187 URasym*), which are clearly indistinguishable from OFoccluded within the current FRET geometry11. 188 To test whether these states are kinetically unresolved by smFRET , we applied three complementary 189 strategies to arrest the OFopen conformation of TmrAB PG: (i) a slow-turnover catalytic mutant 190 (TmrABPG_EQ), (ii) Mg²⁺ depletion using EDTA, and (iii) reverse inhibition by high concentrations of the 191 substrate peptide RRYQKSTEL (R9L) ( Fig. 4). Slow-turnover variants have previously enabled 192 structural separation of OFopen and OF occluded states 11,12,14,25. Mg²⁺ depletion blocks ATP hydrolysis 193 while preserving ATP binding, allowing rapid and reversible trapping 25. We further hypothesized that 194 trans-inhibition by peptide binding sterically restricts PG closure and is therefore expected to 195 stabilize OFopen in a dose-dependent manner26,27. 196 In conditions lacking ATP, either in the absence of nucleotides or in the presence of ADP (3 mM ADP), 197 the slow-turnover variant TmrAB PG_EQ populated a single high-FRET state ( E = 0.97) (Fig. 4a, top and 198 middle). These results indicate that ADP binding alone is insufficient to promote NBD dimerization 199 nor PG opening, consistent with previous biochemical and structural observations 11,12. Upon ATP 200 addition (3 mM ATP), however, the conformational landscape diverged sharply from that of wild-201 type TmrABPG. Instead of the two-state distribution observed for wild-type (apo : E = 0.97, ~20%; ATP-202 bound: E = 0.86, ~80%) (Fig. 2e, bottom), TmrAB PG_EQ exhibited three well-resolved populations with 203 mean E values of E = 0.97 (~14%), E = 0.86 (~55%), and E = 0.63 (~31%) (Fig. 4a, bottom). 204 As in wild-type TmrABPG, the high-FRET population (E = 0.97) corresponds to the IF state, whereas the 205 intermediate-FRET population (E = 0.86) likely represents a dynamic equilibrium of OF open and 206 OFoccluded conformations, as suggested by cryo-EM analyses 11. Post-hydrolysis return states ( URasym 207 and URasym*) are expected to be minimally populated in TmrAB PG_EQ due to its drastically reduced ATP 208 hydrolysis rate12. Notably, the low-FRET ATP-bound population (E = 0.63) was entirely absent in wild-209 type. The transition from E = 0.97 to E = 0.63 corresponds to ΔE = 0.34 and ΔR = 22.5 Å, in close 210 agreement with the IF OFopen distance predicted by AV simulations ( ΔR = 23.2 Å; Fig. 1), thereby 211 postulating E = 0.63 as the OFopen conformation. 212 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 9 Mg²⁺ depletion independently reproduced this three-state landscape. In the absence of Mg 2+, wild-213 type TmrABPG transitioned from a single apo population (without ATP; E = 0.97) (Fig. 4b, top) to three 214 ATP-bound populations (3 mM ATP; E = 0.97, ~13%; E = 0.86, ~56%; E = 0.63, ~31%) ( Fig. 4b, middle) 215 closely resembling those observed for the slow-turnover variant ( Fig. 4a, bottom) . Reintroducing 216 Mg²⁺ abolished the E = 0.63 population and restored the wild-type two-state distribution ( Fig. 4b, 217 bottom). This reversibility confirmed that the ATP-bound OFopen state (E = 0.63) is selectively revealed 218 only when ATP hydrolysis is prevented. 219 Finally, we tested whether periplasmic substrate binding shifts the conformational equilibrium of 220 wild-type TmrAB PG toward OFopen. In the presence of ATP (3 mM), increasing concentrations of 221 peptide substrate (0.3–2 mM R9L) progressively enriched the E = 0.63 population from ~20% to ~38% 222 (Fig. 4c). This dose-dependent stabilization mirrors trans-inhibition behavior reported for human 223 TAP1/2 and reflects the upper substrate-loading capacity of the transporter26. 224 Distance changes derived from smFRET closely match AV simulations, cryo-EM structures (PDB 6RAH, 225 6RAN)11, and DEER/PELDOR measurements 14 (Fig. 4d), together validating assignment of the E = 0.63 226 population as the OFopen conformation. 227 228 Kinetics and thermodynamics of the transport cycle 229 ALEX-smFRET data were acquired with an effective temporal resolution of 2 00 ms (100 ms per 230 excitation channel). Shorter integration times compromised the signal-to -noise ratio and precluded 231 reliable FRET determination . To quantify conformational dynamics, we applied Hidden Markov 232 Modeling (HMM) using MASH-FRET 38, classifying traces as either static (single FRET state) or dynamic 233 (multiple states). Approximately 95% of traces in each condition were classified as static, indicating 234 that most conformational transitions occur at or below our temporal resolution. 235 Although individual transitions could not be directly resolved, population-based analysis ( Fig. 3), 236 combined with biochemical turnover measurements ( Fig. 1–Fig. S1c), allowed estimation of ATP-237 bound dwell times. At saturating ATP conditions well above the apparent Kd, ATP (3 mM, 40 ˚C), wild-238 type TmrAB exhibited a catalytic turnover rate of kcat = 2.57 ± 0.38 s-1 (Fig. 1–Fig. S1c), corresponding 239 to a full transport cycle time ( τcycle) of 395 ± 55 ms. ATP-bound dwell times ( τd) were derived from 240 population ratios obtained from Gaussian fits of the FRET efficiency histograms ( Fig. 3; see Methods, 241 Eq. 3 and Eq. 4). These analyses yielded ATP-bound dwell times of 304 ± 43 ms for TmrAB NBD (~77% 242 ATP-bound) and 316 ± 44 ms for TmrAB PG (~80% ATP-bound), with the remaining ATP-free intervals 243 (~20–23%) accounting for 91 ms and 79 ms of the cycle, respectively. 244 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 10 Together, these measurements establish a quantitative, single-molecule description of conformation 245 state occupancies and dwell times throughout the catalytic cycle of a heterodimeric ABC transporter 246 under active turnover conditions, providing valuable insights into the dynamic landscape of its 247 translocation cycle. 248 249

250 smFRET has become an indispensable tool for dissecting conformational dynamics of membrane 251 proteins, including receptors, ion channels, and transporters, by directly linking structural transitions 252 to functional states With the ABC transporter family, however, smFRET studies have largely been 253 confined to monomeric or homodimeric systems 22-24, leaving the dynamic behavior of asymmetric, 254 heterodimeric transporter comparatively unexplored. Here, we apply smFRET to the heterodimeric 255 type IV ABC transporter TmrAB, extending single-molecule analysis to an asymmetric transporter 256 system and uncovering dynamic features of the transport cycle that are inaccessible to ensemble-257 averaged approaches. 258 By positioning FRET reporters at the nucleotide-binding domains (NBDs) and periplasmic gate (PG), 259 we directly monitored ATP-dependent coupling between chemical energy input and global 260 conformational rearrangements. Importantly, these structural rearrangements are not strictly 261 correlated: NBD dimerization can give rise to either an outward-facing open (OF open) or occluded 262 (OFoccluded) conformation11,12. This decoupling underscores the need to monitor both cytosolic and 263 periplasmic regions to resolve the transport mechanism. 264 Labeling sites previously validated for PELDOR/DEER spectroscopy 10,14,15 were adapted for smFRET 265 and rigorously benchmarked using accessible-volume (AV) simulations 30, fluorescence lifetime 266 analysis, and ensemble FRET titrations. This additional validation is essential because fluorophores 267 impose stricter steric and rotational constraints than nitroxide spin labels 32. Collectively, these 268 controls demonstrate that fluorophore attachment preserves native-like conformational behavior 269 and ATP binding, establishing TmrAB as a robust system for quantitative single-molecule analysis. 270 Consistent with this conclusion, fluorescence lifetime analysis showed no evidence for substantial 271 protein-fluorophore quenching or restricted dye motion, as indicated by donor lifetime shortening 272 and prolonged acceptor lifetimes characteristic of efficient energy transfer17. 273 Single-molecule measurements resolved two dominant FRET populations corresponding to apo and 274 ATP-bound states for both reporter variants TmrAB NBD and TmrAB PG. ATP titrations spanning 275 concentrations well below the reported apparent Kd, ATP (~100 µM for TmrA E523QB)12 up to 276 physiologically relevant levels (3 mM ATP) revealed gradual, concentration-dependent shifts 277 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 11 between these populations, demonstrating the high sensitivity of the FRET constructs to ATP binding. 278 Notably, apparent Kd, ATP values derived from smFRET (2 –13 µM) were substantially lower than those 279 obtained from ensemble FRET measurements (50 –100 µM). This difference likely reflects the 280 inherent selectively of single-molecule analyses for properly folded and catalytically competent 281 transporters. In smFRET experiments, aggregated or inactive species can be identified and excluded 282 during trace selection based on fluorescence intensity, stoichiometry, and photobleaching behavior, 283 thereby enriching the analyzed population for functional molecules. 284 Crucially, the ATP-bound population may reflect a rapidly interconverting ensemble of 285 conformations, rather than a single static structure, potentially including OFopen, OFoccluded, and post-286 hydrolysis unlocked return states (UR asym and UR asym*). These transitions occur faster than the 287 ~200 ms temporal resolution of our measurements, resulting in averaged FRET efficiencies under 288 turnover conditions. Using three independent trapping strategies —slow-turnover catalysis, Mg²⁺ 289 depletion, and substrate trans-inhibition— we stabilized and directly resolved a previously hidden 290 OFopen conformation. The associated distance changes are generally consistent with cryo-EM 291 structures11, PELDOR/DEER data 10,14,15, and simulation-based predictions, suggesting that detergent-292 solubilized TmrAB samples a largely native-like conformational landscape. 293 Although detergent-solubilized and lipid nanodisc-reconstituted TmrAB exhibit similar global 294 conformational states, the conformational space accessible to attached fluorophores may be 295 differentially influenced by membrane-associated environments 19,39,40. In particular, fluorophores 296 attached near the periplasmic gate may experience steric restrictions due to partial overlap with the 297 membrane region, as suggested by AV simulations. While such effects are negligible under the 298 detergent conditions used here , they should be carefully evaluated in future studies employing 299 membrane-embedded systems. Single-molecule measurements further revealed that addition of the 300 peptide substrate induces concentration-dependent shifts in the conformational equilibrium. 301 Increasing substrate concentrations progressively stabilized the OF open state, consistent with trans-302 inhibition behavior observed in human TAP 1/226 and bovine ABCC1 27, and reflecting the finite 303 substrate-loading capacity of the transporter26. 304 Quantitative deconvolution of FRET populations enabled direct determination of conformational 305 state occupancies under physiological ATP concentrations. During active turnover, TmrAB populates 306 the IF state (~20%), the OFopen state (~25%), and OF occluded/post-hydrolysis states (UR asym and URasym*) 307 (~55%) (Fig. 5). The current reporter geometries do not allow direct discrimination between OFoccluded 308 and post-hydrolysis states because fluorophores were placed at the noncanonical nucleotide-binding 309 site. However, contributions from post-hydrolysis states are expected to be minimal for slow-310 turnover TmrAB variant (TmrAB PG_EQ), owing to its drastically reduced ATP hydrolysis rate. To our 311 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 12 knowledge, this represents the first single-molecule quantification of conformational equilibria for a 312 heterodimeric ABC transporter under catalytic conditions. While these distributions broadly align 313 with cryo-EM particle classifications 11,25, smFRET resolves fewer IF states than the cryo-EM 314 distinction between IF wide and IF narrow, and additionally captures ATP-bound intermediates that are 315 challenging to resolve structurally due to rapid interconversion. 316 All smFRET measurements were performed at 40 ˚C to maintain consistency with prior biochemical 317 studies and ensure fluorophore stability. At the physiological temperature of T. thermophilus (68 ˚C), 318 absolute rates of ATP turnover and conformational transitions are expected to increase, although 319 relative state occupancies may remain conserved if the underlying free-energy landscape is 320 preserved. Despite a substantial fraction of static single-molecule trajectories, ATP-dependent 321 population shifts and catalytic rates indicate that TmrAB operates near the temporal resolution limit 322 of our measurements. Integrating smFRET-derived state occupancies with biochemical turnover rates 323 yields an ATP-bound dwell time of approximately 300 ms, in good agreement with previous 324 biochemical estimates9,12. 325 Emerging microsecond-resolution smFRET approaches offer the potential to direct ly visualize short-326 lived intermediates within the transport cycle 41. Future studies could further benefit from three- or 327 four-color FRET strategies 42,43, which would allow simultaneous monitoring of multiple structural 328 elements. In particular, dual labeling of the NBDs and PG could provide direct detection of the 329 OFoccluded state, while probes placed at both canonical and noncanonical nucleotide-binding sites 330 could capture post-hydrolysis conformational dynamics. 331 In summary , this work establish es smFRET as a powerful approach for mapping the dynamic 332 landscape of asymmetric ABC transporters. By quantitatively linking ATP binding, conformational 333 equilibria, and kinetics at the single -molecule level, our study resolved an important aspect of the 334 transport mechanism how chemical energy is transduced into directional transport in heterodimeric 335 ABC systems. Integration of native lipid environments, higher temporal resolution, and substrate 336 engagement will further illuminate the coordination of ATP hydrolysis and substrate translocation 337 during transport. 338 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 13

339 Expression, purification, and labeling of TmrAB 340 His10-tagged TmrAB variants were expressed in E. coli BL21(DE3) (Thermo Fisher Scientific) as 341 described previously 12. Cells were grown in high-salt LB media (Carl Roth) supplemented with 342 100 μg ml-1 ampicillin (PAA Laboratories) at 37 ˚C. At an OD 600 of 0.5, expression was induced with 343 1 mM isopropyl β -D-thiogalactopyranoside (IPTG; Carl Roth), and cultures were incubated for 3 h at 344 37 ˚C. Cells were harvested by centrifugation (4,500 × g, 4 ˚C, 15 min) and stored at –80 ˚C. 345 For purification, cell pellets were resuspended in lysis buffer (20 mM HEPES-NaOH pH 7.5, 300 mM 346 NaCl, 50 µg ml-1 lysozyme, 0.2 mM phenylmethylsulfonyl fluoride (PMSF)) and lysed by sonication. 347 Cell debris was removed by centrifugation (18,000 × g, 4 ˚C, 35 min), and membranes were collected 348 by ultracentrifugation (100,000 × g, 4 ˚C, 30 min). Membranes were solubilized for 2 h at 4 ˚C in 349 purification buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl) containing 20 mM n- dodecyl β-D-350 maltoside (β-DDM; Carl Roth). After ultracentrifugation (100,000 × g, 30 min, 4 ˚C), the supernatant 351 was incubated with Ni- NTA agarose (Bio-Rad) for 1 h at 4 ˚C. The resin was washed with 20 column 352 volumes of wash buffer (20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 1 mM β -DDM) containing 353 50 mM imidazole, and TmrAB was eluted with elution buffer (20 mM HEPES-NaOH pH 7.5, 300 mM 354 NaCl, 1 mM β-DDM, 300 mM imidazole). 355 For fluorophore labeling, TmrAB variants were conjugated via maleimide chemistry using LD555 and 356 LD655 (Lumidyne Technologies). Labeling was carried out at a 1:10:10 molar ratio of protein to each 357 dye in elution buffer for 3 h at 4 ˚C. Excess dye was quenched with 2 mM β-mercaptoethanol (Sigma-358 Aldrich), and the labeled protein was buffer-exchanged into size-exclusion chromatography (SEC) 359 buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM β -DDM) using a Zeba Spin Desalting 360 Column (Thermo Fisher Scientific). Unreacted fluorophores were removed by SEC on a Superdex 200 361 Increase 10/300 GL column (Cytiva). Labeling efficiency was determined by analytical SEC (Superdex 362 200 Increase 3.2/300; Cytiva) by monitoring absorbance at 280, 555, and 655 nm. To preserve 363 sample integrity for smFRET measurements, TmrAB was purified and labeled within a single day, 364 stored on ice, and imaged over the following two days. 365 366 Time-correlated single-photon counting (TCSPC) 367 Fluorescence lifetime measurements were performed using a Fluo Time 100 spectrometer 368 (PicoQuant) equipped for time-correlated single-photon counting (TCSPC). Experiments were carried 369 out on labeled TmrAB in SEC buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM β -DDM). 370 LD555 and LD655 were excited at 510 nm and 610 nm, respectively. Emission was collected using a 371 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 14 620/60 nm bandpass filter for LD555 and a BG4 700 nm long-pass filter for LD655. Photon arrival 372 times were accumulated until the TCSPC histogram reached a peak count of 50,000 photons. 373 Fluorescence decay curves were analyzed by fitting mono- or bi-exponential decay models using 374 FluoFit software (PicoQuant) and amplitude weighted average of fluorescence lifetime was 375 calculated. 376 377 Nanobody production and purification 378 The nanobody Nb9F10 S63C was expressed and purified as described previously 11. Briefly, Nb9F10 S63C 379 was produced in E. coli BL21(DE3) cells grown in Terrific Broth (TB; Carl Roth) supplemented with 380 100 μg ml-1 ampicillin at 37 ˚C. At an OD 600 of 0.6, expression was induced with 1 mM IPTG, followed 381 by overnight incubation at 28 ˚C. Cells were harvested by centrifugation (4,500 × g, 4 ˚C, 15 min) and 382 stored at –80 ˚C. For purification, cell pellets were resuspended in nanobody lysis buffer (25 mM 383 HEPES-NaOH pH 7.4, 300 mM NaCl, 15 mM imidazole, 0.5 mM PMSF) and disrupted by sonication. 384 Cell debris was removed by centrifugation (18,000 × g, 4 ˚C, 35 min), and the clarified lysate was 385 applied to Ni-NTA agarose equilibrated in potassium phosphate (KP i) buffer (25 mM KP i pH 6.5, 386 100 mM KCl, and 0.5 mM tris(2-carboxyethyl) phosphine (TCEP)). Bound nanobody was washed with 387 10 column volumes (CV) of KP i buffer and eluted with 8 CV of elution buffer (25 mM KP i pH 6.0, 388 20 mM KCl, 300 mM imidazole, 0.5 mM TCEP). Eluted fractions were pooled and further purified by 389 cation exchange chromatography (CEX) on a HiTrap SP column (Cytiva) using a linear gradient from 390 low-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, 391 500 mM KCl, 0.5 mM TCEP). The purified nanobody was concentrated and buffer-exchanged into 392 nanobody SEC buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl) using Zeba spin desalting columns, 393 followed by SEC (Superdex 200 Increase 10/300 GL; Cytiva). For site-specific conjugation, Nb9F10 S63C 394 was incubated with a biotin-PEG 11-maleimide linker (Sigma-Aldrich) at a 1.2:1 molar ratio of protein 395 to linker in the presence of 0.5 mM TCEP for 2 h at 4 ˚C. Excess linker was removed by desalting on 396 Zeba Spin Desalting Columns, followed by a final SEC step (Superdex 200 Increase 10/300 GL). 397 398 SDS-PAGE 399 The purity of TmrAB samples was assessed by SDS-PAGE. Resolving gels (12%) were prepared using 400 12% (w/v) acrylamide, 0.5 M Tris-HCl (pH 8.8), 0.13% (w/v) SDS, 0.05% (w/v) ammonium persulphate 401 (APS), and 0.25% (v/v) N,N,N’,N’-tetramethylethylenediamine (TEMED). Stacking gels contained 4.3% 402 (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 Gels were used immediately or stored at 4 ˚C for up to 4 weeks. Protein samples were mixed with 4× 404 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 15 SDS loading buffer containing dithiothreitol (DTT; Sigma-Aldrich) and heated at 90 ˚C for 5 min before 405 loading. Electrophoresis was performed at a constant voltage of 120 V using 1× SDS running buffer 406 (25 mM Tris-HCl pH 8.8, 192 mM glycine, 0.1% SDS). Proteins were visualized by staining with 407 InstantBlueTM Protein Stain (Abcam) for 1 h at room temperature with gentle agitation and imaged 408 using a Fusion FX system (Vilber). 409 410 ATPase activity assay 411 The ATPas e activity of β -DDM-solubilized TmrAB wt was quantified using a Malachite Green-based 412 colorimetric assay as described previously 44. Detergent-solubilized TmrAB (0.6 μM) was incubated in 413 ATPase buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 2 mM MgCl 2, 1 mM β-DDM) containing 414 3 mM ATP (Sigma-Aldrich) at 40 ˚C for 7 min. Autohydrolysis controls were prepared by incubation of 415 ATP in ATPase buffer without protein. Reactions were quenched by adding 20 mM H 2SO4, followed 416 by incubation with 3 mM Malachite Green (Thermo Fisher Scientific), 0.2% (v/v) Tween20 (Carl Roth), 417 and 1.5% (w/v) ammonium molybdate (Carl Roth) for 10 min at room temperature. The absorbance 418 at 620 nm was recorded on a CLARIOstar v.5.20 R5 plate reader (BMG LABTECH). 419 420 Ensemble FRET measurements 421 The ATP binding and FRET characteristics of selected TmrAB variants were assessed by ensemble 422 FRET. Labeled TmrAB (100 nM) was incubated with increasing concentrations of ATP at 42 ˚C for 423 5 min. Donor-excited emission was recorded from 550 –700 nm with an excitation wavelength of 424 520 nm using a Clariostar v.5.20 R5 plate reader (BMG LABTECH). Acceptor emission intensities at 425 675 nm were plotted against ATP concentration and fitted with a hyperbolic function to determine 426 the apparent dissociation constant (Kd, ATP) for each variant. 427 428 Functionalization of glass slides for single-molecule FRET analysis 429 Glass coverslips used for TmrAB immobilization in smFRET experiments were functionalized by 430 PEGylation as described previously 45. Coverslips (Carl Roth) were cleaned by sequential sonication in 431 Milli-Q water and analytical-grade acetone (>99.9%; VWR International), followed by oxygen plasma 432 treatment (0.3 mbar, 80% power, 15 min) using a Zepto plasma cleaner (Diener) and a 10 min 433 incubation in methanol (Avantor, Gliwice, PL). Coverslips were then silanized by incubation for 434 30 min in a solution of 100 ml methanol, 5 ml acetic acid, and 3 ml 3-aminopropyltrimethoxysilane 435 (APTES; Tokyo Chemical Industry). After silanization, sides were rinsed four times with methanol and 436 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 16 dried under a nitrogen stream. Surface functionalization was achieved using a mixture of 437 biotinylated-PEG (4 mol%) and nonbiotinylated-PEG (96 mol%; Rapp Polymer). The PEG solution was 438 sandwiched between two coverslips and incubated overnight in a humidity chamber. Coverslips were 439 then rinsed thoroughly with Milli-Q water and dried under nitrogen. To enhance passivation, a 440 second PEGylation step was performed using 25 mM CH 3-PEG-NHS (333 Da; Thermo Fisher Scientific) 441 under the same conditions. Finally, slides were rinsed with Milli-Q water, dried under nitrogen, and 442 stored at –20 ˚C under argon until use. 443 444 Single-molecule FRET imaging 445 Single-molecule FRET (smFRET) experiments were performed using a flow chamber system (Ibidi). 446 Chambers were assembled by placing a biotin-PEG- functionalized glass slide onto a μ -Slide I Luer 447 Family flow channel (Ibidi), with the functionalized surface facing inward. All buffers and Milli-Q 448 water were filtered through 0.2 μm filters (Sigma -Aldrich). Chambers were washed with 1 ml Milli-Q 449 water and incubated with 0.2 mg ml-1 streptavidin (Sigma-Aldrich) at 4 ˚C for 30 min to allow binding 450 to the biotin-PEG surface. Unbound streptavidin was removed by washing with 1 ml Milli-Q water. 451 The surface was then treated with 0.3 mg ml-1 biotinylated-PEG 11-Nb9F10S63C at 4 ˚C for 45 min, 452 followed flushing with 2 ml SEC buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 1 mM β -DDM). 453 Detergent-solubilized, fluorophore-labeled TmrAB (100 nM) was added and incubated at 4 ˚C for 1 h. 454 Unbound protein was removed by five washes with 1 ml TmrAB-SEC buffer. Chambers were 455 equilibrated with 1 ml of imaging buffer containing 25 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 456 3 mM MgCl 2, 50 mM glucose, 5 mM Trolox, 7.5 U ml-1 pyranose oxidase, and 1 kU ml-1 catalase, 457 supplemented with the desired ATP concentration. For EDTA trapping experiments, MgCl 2 was 458 omitted and replaced with 3 mM ethylenediaminetetraacetic acid (EDTA; Sigma-Aldri ch). smFRET 459 data were acquired at 40 ˚C using alternating laser excitation (ALEX) on a total internal reflection 460 fluorescence (TIRF) microscope (NanoImager S, ONI, Oxford, UK). Typically, 600 frames were 461 recorded per region of interest (ROI) with 100 ms exposure time. Laser powers were 0.8 mW cm-2 462 (532 nm) and 0.9 mW cm-2 (640 nm). Data were recorded in 1-min intervals, except for TmrAB NBD 463 variant in apo and 3 mM ATP conditions, where 3-min intervals were used to confirm that 464 conformation transitions do not occur on timescales longer that one minute due to the reduced 465 temperature. 466 467 Single-molecule FRET data analysis 468 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 17 Single-molecule FRET (smFRET) measurements were performed using alternating laser excitation 469 (ALEX), allowing assignment of detected photons based on both excitation and emission 470 wavelengths. Photon counts for each molecule were extracted using NanoImager software (ONI 471 NanoImager, Development Build) and classified into three detection channels: donor excitation with 472 donor emission (𝑓𝐷𝑒𝑥 𝐷𝑒𝑚), donor excitation with acceptor emission ( 𝑓𝐷𝑒𝑥 𝐴𝑒𝑚), and acceptor excitation with 473 acceptor emission (𝑓𝐴𝑒𝑥 𝐴𝑒𝑚). Traces were analyzed with DeepFRET 35 and manually curated. To minimize 474 bias, a second researcher independently curated traces from both ATP-free and 3 mM ATP samples, 475 yielding 98% overlap between curations. FRET efficiency (E) and stoichiometry (S) were calculated as: 476 Population analysis was performed by constructing one-dimensional histograms of FRET efficiency (E) 477 and stoichiometry ( S) using OriginPro 2024 (OriginLab). Histograms were fitted with two Gaussian 478 distributions corresponding to the ATP-free state (defined from apo samples) and the ATP-bound 479 state (defined by a two-component fit at saturating ATP). Hidden Markov Modeling (HMM) of 480 individual traces was performed using MASH-FRET 38 to distinguish dynamic from static molecules 481 within each sample. 482 483 ATP-bound dwell times and distribution of conformational states 484 The ATP-bound dwell time (τd) was estimated as: 485 where fATP-bound is the fraction of ATP-bound molecules derived from Gaussian fits of the FRET 486 efficiency histograms ( Fig. 3). This approach assumes (i) ATP hydrolysis occurs exclusively at the 487 canonical NBS, with negligible contribution from the noncanonical site 9,11,14, and (ii) the majority of 488 molecules are catalytically competent and continuously cycling. 489 The distribution of conformational states within ATP-bound FRET population ( E = 0.86) of TmrAB PG 490 under turnover conditions (3 mM ATP) was determined using a two-state model: 491 𝐸 = 𝑓𝐷𝑒𝑥 𝐴𝑒𝑚 𝑓𝐷𝑒𝑥 𝐷𝑒𝑚 + 𝑓𝐴𝑒𝑥 𝐴𝑒𝑚 Eq. 1 𝑆 = 𝑓𝐷𝑒𝑥 𝐷𝑒𝑚 + 𝑓𝐷𝑒𝑥 𝐴𝑒𝑚 𝑓𝐷𝑒𝑥 𝐷𝑒𝑚 + 𝑓𝐷𝑒𝑥 𝐴𝑒𝑚 + 𝑓𝐴𝑒𝑥 𝐴𝑒𝑚 Eq. 2 𝜏𝑑 = 𝜏𝑐𝑦𝑐𝑙𝑒 × 𝑓𝐴𝑇𝑃−𝑏𝑜𝑢𝑛𝑑 Eq. 3 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 18 where fopen is the fraction of OF open state (E open = 0.63) readily resolved under trapping conditions 492 (Fig. 4), and fother represents the combined fraction of PG-closed states (OF occluded/URasym/URasym*, 493 Eother = 0.97). 494 𝐸 = 𝑓𝑜𝑝𝑒𝑛 × 𝐸𝑜𝑝𝑒𝑛 + 𝑓𝑜𝑡ℎ𝑒𝑟 × 𝐸𝑜𝑡ℎ𝑒𝑟 Eq. 4 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 19

495 1. Davidson AL, Dassa E, Orelle C, Chen J. Structure, function, and evolution of bacterial ATP-496 binding cassette systems. Microbiol Mol Biol Rev 72, 317–364 (2008). 497 2. Rees DC, Johnson E, Lewinson O. ABC transporters: the power to change. Nat Rev Mol Cell Biol 498 10, 218–227 (2009). 499 3. Thomas C, Tampé R. Structural and mechanistic principles of ABC transporters. Annu Rev 500 Biochem 89, 605–636 (2020). 501 4. Locher KP. Mechanistic diversity in ATP-binding cassette (ABC) transporters. Nat Struct Mol Biol 502 23, 487–493 (2016). 503 5. Thomas C , et al. Structural and functional diversity calls for a new classification of ABC 504 transporters. FEBS Lett 594, 3767–3775 (2020). 505 6. Robey RW, Pluchino KM, Hall MD, Fojo AT, Bates SE, Gottesman MM. Revisiting the role of ABC 506 transporters in multidrug-resistant cancer. Nat Rev Cancer 18, 452–464 (2018). 507 7. Abele R, Tampé R. The ABCs of immunology: structure and function of TAP, the transporter 508 associated with antigen processing. Physiology (Bethesda) 19, 216–224 (2004). 509 8. Kim J , et al. Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC 510 exporter. Nature 517, 396–400 (2015). 511 9. Zutz A , et al. Asymmetric ATP hydrolysis cycle of the heterodimeric multidrug ABC transport 512 complex TmrAB from Thermus thermophilus. J Biol Chem 286, 7104–7115 (2011). 513 10. Nöll A , et al. Crystal structure and mechanistic basis of a functional homolog of the antigen 514 transporter TAP. Proc Natl Acad Sci U S A 114, E438–E447 (2017). 515 11. Hofmann S , et al. Conformation space of a heterodimeric ABC exporter under turnover 516 conditions. Nature 571, 580–583 (2019). 517 12. Stefan E, Hofmann S, Tampé R. A single power stroke by ATP binding drives substrate 518 translocation in a heterodimeric ABC transporter. eLife 9, e55943 (2020). 519 13. Stefan E , et al. De novo macrocyclic peptides dissect energy coupling of a heterodimeric ABC 520 transporter by multimode allosteric inhibition. eLife 10, e67732 (2021). 521 14. Barth K, Rudolph M, Diederichs T, Prisner TF, Tampé R, Joseph B. Thermodynamic basis for 522 conformational coupling in an ATP-binding cassette exporter. J Phys Chem Lett 11, 7946 –7953 523 (2020). 524 15. Barth K, Hank S, Spindler PE, Prisner TF, Tampé R, Joseph B. Conformational coupling and trans-525 inhibition in the human antigen transporter ortholog TmrAB resolved with dipolar EPR 526 spectroscopy. J Am Chem Soc 140, 4527–4533 (2018). 527 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 20 16. Agam G, et al. Reliability and accuracy of single-molecule FRET studies for characterization of 528 structural dynamics and distances in proteins. Nat Methods 20, 523–535 (2023). 529 17. Hellenkamp B , et al. Precision and accuracy of single-molecule FRET measurements-a multi-530 laboratory benchmark study. Nat Methods 15, 669–676 (2018). 531 18. Sasmal DK, Pulido LE, Kasal S, Huang J. Single-molecule fluorescence resonance energy transfer 532 in molecular biology. Nanoscale 8, 19928–19944 (2016). 533 19. Bartels K, Lasitza-Male T, Hofmann H, Löw C. Single-molecule FRET of membrane transport 534 proteins. ChemBioChem 22, 2657–2671 (2021). 535 20. Lerner E , et al. FRET-based dynamic structural biology: Challenges, perspectives and an appeal 536 for open-science practices. eLife 10, e60416 (2021). 537 21. Nettels D, Galvanetto N, Ivanović MT, Nüesch M, Yang T, Schuler B. Single -molecule FRET for 538 probing nanoscale biomolecular dynamics. Nat Rev Phys 6, 587–605 (2024). 539 22. Wang L, Johnson ZL, Wasserman MR, Levring J, Chen J, Liu S. Characterization of the kinetic cycle 540 of an ABC transporter by single-molecule and cryo-EM analyses. eLife 9, e56451 (2020). 541 23. Levring J, Terry DS, Kilic Z, Fitzgerald G, Blanchard SC, Chen J. CFTR function, pathology and 542 pharmacology at single-molecule resolution. Nature 616, 606–614 (2023). 543 24. Husada F , et al. Conformational dynamics of the ABC transporter McjD seen by single-molecule 544 FRET. EMBO J 37, e100056 (2018). 545 25. Nocker C, Pečak M, Nocker T, Fahim A, Sušac L, Tampé R. Single -molecule dynamics reveal ATP 546 binding alone powers substrate translocation by an ABC transporter. BioRxiv, 547 2025.2011.2027.690960 (2025). 548 26. Grossmann N, Vakkasoglu AS, Hulpke S, Abele R, Gaudet R, Tampé R. Mechanistic determinants 549 of the directionality and energetics of active export by a heterodimeric ABC transporter. Nat 550 Commun 5, 5419 (2014). 551 27. Sun P , et al. Substrate recognition diversity and transport dynamics of ABCC1. Nat Commun 16, 552 10499 (2025). 553 28. Altman RB , et al. Cyanine fluorophore derivatives with enhanced photostability. Nat Methods 9, 554 68–71 (2011). 555 29. Martin MI , et al. Leveraging Baird aromaticity for advancement of bioimaging applications. J Phys 556 Org Chem 36, (2023). 557 30. Kalinin S , et al. A toolkit and benchmark study for FRET-restrained high-precision structural 558 modeling. Nat Methods 9, 1218–1225 (2012). 559 31. Ha T, Tinnefeld P. Photophysics of fluorescent probes for single-molecule biophysics and super-560 resolution imaging. Annu Rev Phys Chem 63, 595–617 (2012). 561 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 21 32. Sindbert S , et al. Accurate distance determination of nucleic acids via Förster resonance energy 562 transfer: implications of dye linker length and rigidity. J Am Chem Soc 133, 2463–2480 (2011). 563 33. Dale RE, Eisinger J, Blumberg WE. The orientational freedom of molecular probes. The 564 orientation factor in intramolecular energy transfer. Biophys J 26, 161–193 (1979). 565 34. van der Meer BW. Kappa-squared: from nuisance to new sense. J Biotechnol 82, 181–196 (2002). 566 35. Thomsen J , et al. DeepFRET, a software for rapid and automated single-molecule FRET data 567 classification using deep learning. eLIfe 9, e60404 (2020). 568 36. Hohlbein J, Craggs TD, Cordes T. Alternating-laser excitation: single-molecule FRET and beyond. 569 Chem Soc Rev 43, 1156–1171 (2014). 570 37. Asher WB , et al. Single-molecule FRET imaging of GPCR dimers in living cells. Nat Methods 18, 571 397–405 (2021). 572 38. Hadzic M, Borner R, Konig SLB, Kowerko D, Sigel RKO. Reliable state identification and state 573 transition detection in fluorescence intensity -based single -molecule Förster Resonance Energy -574 Transfer data. J Phys Chem B 122, 6134–6147 (2018). 575 39. Dimura M, Peulen TO, Hanke CA, Prakash A, Gohlke H, Seidel CA. Quantitative FRET studies and 576 integrative modeling unravel the structure and dynamics of biomolecular systems. Curr Opin 577 Struct Biol 40, 163–185 (2016). 578 40. Lam K, Tajkhorshid E. Membrane interactions of Cy3 and Cy5 fluorophores and their effects on 579 membrane-protein dynamics. Biophys J 119, 24–34 (2020). 580 41. Grabenhorst L, Sturzenegger F, Hasler M, Schuler B, Tinnefeld P. Single-molecule FRET at 10 MHz 581 count rates. J Am Chem Soc 146, 3539–3544 (2024). 582 42. Bonhomme L , et al. Triple labeling resolves a GPCR intermediate state by using three-color single 583 molecule FRET. J Am Chem Soc 147, 17689–17700 (2025). 584 43. Yim SW , et al. Four-color alternating-laser excitation single-molecule fluorescence spectroscopy 585 for next-generation biodetection assays. Clin Chem 58, 707–716 (2012). 586 44. Diederichs T, Tampé R. Single cell-like systems reveal active unidirectional and light-controlled 587 transport by nanomachineries. ACS Nano 15, 6747–6755 (2021). 588 45. Chandradoss SD, Haagsma AC, Lee YK, Hwang JH, Nam JM, Joo C. Surface passivation for single-589 molecule protein studies. J Vis Exp, 50549 (2014). 590 46. Chaptal V , et al. Substrate-bound and substrate-free outward-facing structures of a multidrug 591 ABC exporter. Sci Adv 8, eabg9215 (2022). 592 593 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 22 Author contributions 594 M.P. prepared all TmrAB samples and carried out the experiments for this study. M.P. performed 595 data analysis. Curated traces were independently checked by C.N. to avoid human bias. M.P. and 596 C.N. prepared functionalized glass slides for single-molecule FRET . M.P. and R.T. wrote the 597 manuscript. R.T. conceived and supervised the work. 598 599

600 This work was supported by the European Research Council (ERC Advanced Grant 101141396 to 601 R.T.), the German Research Foundation via the Collaborative Research Center CRC 1507/P18 to R.T. 602 and the Research Training Group (GRK 1986/B4.7 to R.T.). We thank Jan F.M. Stuke and Jonas 603 Göhmann for their support in automating trace extraction from ONI NanoImager software, Dr. David 604 Glück for guidance on lifetime measurements, and Tobias Nocker for preparing nanobodies used in 605 the study. We are also grateful to the Wachtveitl lab (Goethe University Frankfurt) for access to their 606 FluoTime 100 spectrometer (PicoQuant). Finally, we thank Dr. Rupert Abele, Dr. David Glück, Dr. 607 Simon Trowitzsch, Inga Nold, and Andrea Pott for helpful comments on the manuscript and 608 proofreading. 609 610 Data and materials availability 611 All data are available in the main text or the supplementary materials. All other data are available 612 from the corresponding author upon reasonable request. Source data are provided with this paper: 613 DOI: 614 https://doi.org/10.25716/gude.0jbq-k1q6. 615 616 Competing Interest 617 The authors declare no competing interest. 618 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 23 Figures 619 620 Figure 1. Accessible-volume (AV) simulations of LD fluorophores on TmrAB variants. AV simulations 621 were performed for LD555 (donor) and LD655 (acceptor) fluorophores attached to the selected 622 TmrAB labeling sites to assess whether donor-acceptor distances are suitable for smFRET 623 measurements30. a, TmrAB NBD (TmrA C416BL458C) and b, TmrABPG (TmrA C416A, T61C BR56C) in the inward-624 facing wide ( IFwide; PDB: 6RAN, left) and outward-facing open ( OFopen; PDB: 6RAH, right) 625 conformations. Approximate membrane position is indicated by the dashed grey line. For all 626 simulations, TmrA is shown in blue with LD655 (orange) and TmrB in yellow with LD555 (green). c, AV 627 simulations confirmed that donor-acceptor distances ( RDA) remain within the FRET-sensitive range in 628 both conformations, predicting measurable shifts in simulated FRET efficienc ies ( Esim). Cryo-EM 629 structures of TmrAB reconstituted in lipid nanodiscs 11 were used as templates . Structures were 630 determined either in apo state (apo) or in presence of 3 mM ATP (turnover) . Outward-facing open 631 (OFopen) and outward-facing occluded ( OFoccluded) structures were obtained via orthovanadate 632 trapping (Vi) or by using the slow-turnover catalytic mutant TmrAE523QB (EQ). 633 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 24 634 Figure 2. ATP-induced conformational changes of TmrAB analyzed by smFRET. a, Experimental 635 setup. TmrABNBD (left) and TmrAB PG (right) variants were labeled with LD555/LD655 and tethered to 636 PEGylated coverslips via a biotinylated anti-TmrB nanobody ( Nb9F10S63C). b, smFRET imaging. 637 Samples were recorded using total internal reflection fluorescence (TIRF) microscopy with alternating 638 laser excitation (ALEX ; donor: 532 nm; acceptor: 640 nm). Emission was collected in two channels 639 (donor: 498-620 nm; acceptor: 662-710 nm) . Donor fluorescence ( 𝑓𝐷𝑒𝑥 𝐷𝑒𝑚: donor excitation at donor 640 emission), sensitized acceptor fluorescence ( 𝑓𝐷𝑒𝑥 𝐴𝑒𝑚: donor excitation at acceptor emission), and direct 641 acceptor fluorescence ( 𝑓𝐴𝑒𝑥 𝐴𝑒𝑚: acceptor excitation at acceptor emission) were used to calculate FRET 642 efficiency ( E) and stoichiometry ( S). Fluorescence time traces were analyzed with DeepFRET 35. 643 c, Representative TmrAB NBD trace in ATP-bound state (top) and corresponding FRET 644 efficiency/stoichiometry plot (bottom). Donor emission upon donor excitation is shown in green as 645 the number of detected photons per frame (counts), acceptor intensity upon donor excitation in 646 orange, FRET efficiency ( E) in black, and stoichiometry ( S) in grey. The slight increase in donor 647 fluorescence observed during the first seconds of acquisition reflects photophysical equilibration of 648 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 25 the fluorophore and instrumental stabilization and does not affect FRET efficiency or stoichiometry, 649 which are ratio-based and remain constant over time 17. d,e, Population analysis. FRET efficiency (E ) 650 histograms for (d) TmrABNBD and (e) TmrABPG are shown for the apo state (top) and ATP-bound state 651 (bottom; 3 mM ATP ). Histograms were fitted with two Gaussian populations corresponding to the 652 apo state (blue; defined from apo measurements) and the ATP-bound state (orange; defined from 653 two-component fits at saturati ng ATP). Dotted vertical lines indicate mean E values of each 654 population. Population fractions , calculated from Gaussian areas, are summarized schematically in 655 each panel. 656 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 26 657 Figure 3. ATP-dependent shifts in smFRET populations of TmrAB. a,c, Increasing ATP concentrations 658 gradually redistributed the population between apo and ATP-bound conformations for ( a) TmrABNBD 659 and ( c) TmrAB PG. FRET efficiency ( E) histograms were fitted with two Gaussian populations 660 corresponding to the ATP-free state ( blue: defined from apo samples) and the ATP-bound state 661 (orange; determined from fits at saturating ATP). Dotted vertical lines indicate the mean E values of 662 each population. Relative proportion fractions, calculated from Gaussian areas, are summarized 663 schematically in each panel. b,d, ATP-binding curves were obtained by plotting the fraction of 664 molecules in the ATP-bound states as a function of ATP concentration for ( b) TmrAB NBD (reporting 665 NBD dimerization) and ( d) TmrAB PG (reporting PG opening). Data were fitted with a Langmuir 666 isotherm to determine the apparent dissociation constant Kd, ATP of each variant. 667 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 27 668 Figure 4. Iden tification of the outward-facing open ( OFopen) conformation. a–c, Three 669 complementary approaches were employed to resolve OFopen state: ( a) the slow-turnover variant 670 TmrABPG_EQ, (b) imaging in Mg2+-free buffer supplemented with EDTA, and ( c) stabilizing via reverse 671 inhibition using high concentrations of peptide substrate. FRET efficiency ( E) histograms were fitted 672 with three Gaussian populations corresponding to the ATP-free state (blue) , the ATP-bound state 673 (orange), and the stabilized OF open state (green) . All three strategies revealed a distinct OFopen 674 population. Dotted vertical lines indicate the mean E values of each population. Relative proportion 675 fractions, calculated from Gaussian areas, are summarized schematically in each panel. d, 676 Comparison of inter-residues distances. Distances (Å) between selected residues on the NBDs and PG 677 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 28 of TmrAB were determined by multiple methods: smFRET values (this study) for detergent-678 solubilized TmrAB; c ryo-EM distances (C β–Cβ) from nanodisc-reconstituted TmrAB (PDB 6RAH, 679 6RAN)11; accessible-volume (AV) simulation distances for nanodisc-reconstituted TmrAB (this study) ; 680 and PELDOR/DEER distances from detergent-solubilized TmrAB15. 681 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 29 682 Figure 5. Conformational state distribution and catalytic cycle of TmrAB under active turnover. 683 Schematic of the TmrAB transport cycle summarizing major conformational states and their 684 estimated population distributions under physiological ATP concentrations (3 mM, 40 ˚C). a, The 685 inward-facing apo state (IF narrow and IF wide; blue arc) accounts for ~20% of molecules and is 686 characterized by separated NBDs and a cytosol-accessible substrate-binding cavity. Substrate binding 687 stabilizes the IFwide conformation11. Independent of substrate, ATP binding induces NBD dimerization 688 and transition to the ATP-bound ensemble. b,c, Under substrate-bound turnover conditions, TmrAB 689 proceeds via the ( b) OFoccluded to (c) OFopen state in which the substrate release occurs . Under steady-690 state turnover, the ATP-bound ensemble rapidly interconverts between substrate-free OFoccluded and 691 OFopen accounting for ~25% of the ATP-bound population (green circle). These transitions occur faster 692 than the ~200 ms temporal resolution of standard smFRET measurement, resulting in an averaged 693 signal under turnover conditions. OF occluded likely serves as an obligate intermediate between IF and 694 OFopen, preventing substrate backflow by maintaining a substrate-binding cavity occluded during the 695 structural rearrangements of the PG and NBDs. Although a substrate-bound OF occluded state has not 696 been directly observed for TmrAB, its existence is supported by structures of the homodimeric 697 type IV transporter BmrA 46. Reduced ATP hydrolysis or substrate trans-inhibition enables trapping of 698 the transporter in the OFopen. state. d,e, ATP hydrolysis and phosphate (P i) release generate post-699 hydrolysis return states: ( d) URasym and ( e) URasym*. Subsequent ADP release restores the apo IF 700 conformation, completing the transport cycle. Overall, the ATP-bound phase ( b–e) represents ~55% 701 occupancy (orange arc) with an estimated dwell time of ~310 ms, whereas the apo/ATP-rebinding 702 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 30 phase (a) lasts ~90 ms, yielding a total cycle time of ~400 ms ( kcat = 2.57 s⁻¹). TmrA is shown in blue, 703 TmrB in yellow, substrate as a green diamond, and nucleotides as orange symbols. Dotted grey boxes 704 indicate the approximate position of the NBD dimer interface. 705 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 31 Figure supplements 706 707 Figure 1–Figure supplement 1. Quality of TmrAB purification and fluorophore labeling. a, SDS-PAGE 708 analysis (10%, reducing conditions, Coomassie staining) of successive purification steps: M, molecular 709 weight marker; L, cell lysate; B, Ni-NTA beads after incubation with lysate; FT, flow-through; W, 710 wash; E, eluted TmrAB ; BE, TmrAB after buffer exchange; C, concentrated TmrAB; CFT, concentrator 711 flow-through; 1 and 2, first and second peaks eluted from size-exclusion chromatography (SEC). Only 712 the second peak was used for subsequent FRET experiments . b, SEC (Superdex 200 increase 10/300 713 GL) confirming monodispersity of labeled TmrAB and efficient removal of free fluorophores . 714 Representative chromatogram is shown for TmrAB PG. c, ATP hydrolysis activity of purified wild-type 715 TmrAB ( 60 nM TmrAB wt) measured at 40 ˚C for 7 min. Released inorganic phosphate (P i) was 716 quantified using the Malachite Green assay. Data were fitted to a Michaelis-Menten model, yielding 717 Km = 0.97 ± 0.28 mM and kcat = 2.57 ± 0.38 s-1. d–f, Analytical SEC (Superdex 200 increase 3.2/300) 718 used to determine fluorophore labeling efficienc ies for each variant: ( d) TmrABNBD, LD555: ~ 55%, 719 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 32 LD655: ~ 53%; ( e) TmrAB PG, LD555: ~ 43%, LD655: ~52%; and (f ) TmrAB PG_EQ, LD555: ~ 42%, LD655: 720 ~52%. 721 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 33 722 Figure 1–Figure supplement 2. FRET capabilities of labeled TmrAB variants. a, Time-correlated 723 single-photon counting histograms of LD555 (left) and LD655 (middle ) measured under three 724 conditions: free dye in buffer (black), LD555/LD655-labeled TmrAB NBD (orange), and LD555/LD655-725 labeled TmrAB PG (blue). Amplitude-weighted average fluorescence lifetimes are summarized in the 726 table (right), confirming sufficient rotational freedom for reliable FRET measurements . b–d, 727 Ensemble donor-exited emission spectra (550 –700 nm, excitation 520 nm) of ( b) TmrAB NBD, ( c) 728 TmrABPG, and (d) the slow-turnover variant TmrAB PG_EQ, stochastically labeled with LD555/LD655 and 729 incubated with increasing ATP concentrations. Spectra are normalized to donor intensity in the apo 730 state. ATP-dependent donor quenching and acceptor sensitization indicate that all variants retain 731 FRET capability. e–g, Fractional fluorescence change s, ( F-F₀)/F₀, where F is acceptor emission 732 intensity and F₀ is the intensity in the apo state, plotted as a function of ATP concentration for ( e) 733 TmrABNBD, ( f) TmrAB PG, and ( g) TmrAB PG_EQ. Data were fitted with a hyperbolic binding model to 734 determine apparent Kd, ATP values, consistent with ensemble FRET measurements of ATP binding. 735 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 34 736 Figure 2–Figure supplement 1. Representative smFRET traces of TmrAB NBD. Representative single-737 molecule FRET (smFRET) traces of TmrAB NBD were recorded ( a) in the ATP-free state and ( b, c) in the 738 presence of 3 mM ATP. Hidden Markow modeling (HMM) was applied to classify traces into (b ) static 739 and (c) dynamic, based on the absence or presence of transitions between ATP-free and ATP-bound 740 conformational states. Donor fluorescence intensity upon donor excitation is shown in green, 741 acceptor fluorescence intensity upon donor excitation in orange , FRET efficiency ( E) in black, and 742 stoichiometry (S) in grey. 743 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint 35 744 Figure 2–Figure supplement 2. Representative smFRET traces of TmrAB PG. Representative single-745 molecule FRET (smFRET) traces of TmrAB PG were recorded (a ) in the ATP-free state and ( b, c) in the 746 presence of 3 mM ATP. Hidden Markow modeling (HMM) was applied to classify traces into ( b) static 747 and (c) dynamic, based on the absence or presence of transitions between ATP-free and ATP-bound 748 conformational states. Donor fluorescence intensity upon donor excitation is shown in green, 749 acceptor fluorescence intensity upon donor excitation in orange, FRET efficiency ( E) in black, and 750 stoichiometry (S) in grey. 751 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted February 16, 2026. ; https://doi.org/10.64898/2026.02.12.705656doi: bioRxiv preprint