The conformational dynamics in ABC transporters is largely elusive. The ABC importer GlnPQ from Lactococcus lactis has different covalently linked substrate-binding domains (SBDs), thus making it an excellent model system to elucidate the dynamics and role of the SBDs in transport. We demonstrate by single-molecule spectroscopy that the two SBDs intrinsically transit from open to closed ligand-free conformation, and the proteins capture their amino acid ligands via an induced-fit mechanism. High-affinity ligands elicit transitions without changing the closed-state lifetime, whereas low-affinity ligands dramatically shorten it. We show that SBDs in the closed state compete for docking onto the translocator, but remarkably the effect is strongest without ligand. We find that the rate-determining steps depend on the SBD and the amino acid transported. We conclude that the lifetime of the closed conformation controls both SBD docking to the translocator and substrate release.

Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch from an open to a closed conformation upon substrate binding, providing specificity for transport. We investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs sample a range of conformations that activate transport. Certain non-transported ligands leave the structure largely unaltered or trigger a conformation distinct from that of transported substrates. Intriguingly, in some cases, similar SBP conformations are formed by both transported and non-transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP interactions, SBP conformational dynamics and substrate transport.

Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.

ABC transporters utilize ATP for export processes to provide cellular resistance against toxins, antibiotics, and harmful metabolites in eukaryotes and prokaryotes. Based on static structure snapshots, it is believed that they use an alternating access mechanism, which couples conformational changes to ATP binding (outward‐open conformation) and hydrolysis (inward‐open) for unidirectional transport driven by ATP. Here, we analyzed the conformational states and dynamics of the antibacterial peptide exporter McjD from Escherichia coli using single‐molecule Förster resonance energy transfer (smFRET). For the first time, we established smFRET for an ABC exporter in a native‐like lipid environment and directly monitor conformational dynamics in both the transmembrane‐ (TMD) and nucleotide‐binding domains (NBD). With this, we unravel the ligand dependences that drive conformational changes in both domains. Furthermore, we observe intrinsic conformational dynamics in the absence of ATP and ligand in the NBDs. ATP binding and hydrolysis on the other hand can be observed via NBD conformational dynamics. We believe that the progress made here in combination with future studies will facilitate full understanding of ABC transport cycles.

ATP-binding cassette (ABC) transporters play crucial roles in cellular processes, such as nutrient uptake, drug resistance, cell-volume regulation and others. Despite their importance, all proposed molecular models for transport are based on indirect evidence, i.e. functional interpretation of static crystal structures and ensemble measurements of function and structure. Thus, classical biophysical and biochemical techniques do not readily visualize dynamic structural changes. We recently started to use single-molecule fluorescence techniques to study conformational states and changes of ABC transporters in vitro, in order to observe directly how the different steps during transport are coordinated. This review summarizes our scientific strategy and some of the key experimental advances that allowed the substrate-binding mechanism of prokaryotic ABC importers and the transport cycle to be explored. The conformational states and transitions of ABC-associated substrate-binding domains (SBDs) were visualized with single-molecule FRET, permitting a direct correlation of structural and kinetic information of SBDs. We also delineated the different steps of the transport cycle. Since information in such assays are restricted by proper labelling of proteins with fluorescent dyes, we present a simple approach to increase the amount of protein with FRET information based on non-specific interactions between a dye and the size-exclusion chromatography (SEC) column material used for final purification.

Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of dsDNA following its interaction with unlabelled proteins (BamHI,

25 proteins, solute translocation, substrate transport 26 27 2 ABSTRACT 28 Substrate-binding proteins (SBPs) are associated with ATP-binding cassette importers and switch 29from an open-to a closed-conformation upon substrate binding providing specificity for transport. We 30 investigated the effect of substrates on the conformational dynamics of six SBPs and the impact on 31 transport. Using single-molecule FRET, we reveal an unrecognized diversity of plasticity in SBPs. We 32 show that a unique closed SBP conformation does not exist for transported substrates. Instead, SBPs 33 sample a range of conformations that activate transport. Certain non-transported ligands leave the 34 structure largely unaltered or trigger a conformation distinct from that of transported substrates. 35Intriguingly, in some cases similar SBP conformations are formed by both transported and non-36 transported ligands. In this case, the inability for transport arises from slow opening of the SBP or the 37 selectivity provided by the translocator. Our results reveal the complex interplay between ligand-SBP 38 interactions, SBP conformational dynamics and substrate transport. 39 3 INTRODUCTION 40 ATP-binding cassette (ABC) transporters facilitate the unidirectional trans-bilayer movement of a 41 diverse array of molecules using the energy released from ATP hydrolysis 1 . ABC transporters share a 42 common architecture, with the translocator unit comprising two transmembrane domains (TMDs) that 43 form the translocation pathway and two cytoplasmic nucleotide-binding domains (NBDs) that bind 44 and hydrolyse ATP. ABC importers require an additional extra-cytoplasmic accessory protein referred 45 to as a substrate-binding protein SBP or domain SBD (hereafter SBDs and SBPs are both termed 46 SBPs) 2-4 . ABC importers that employ SBPs can be subdivided as Type I or Type II based on structural 47 and mechanistic distinctions 5, 6 . A unifying feature of the transport mechanism of Type I and Type II 48 ABC importers is the binding and delivery of substrate from a dedicated SBP to the translocator unit 49 for import into the cytoplasm. 50 Bacterial genomes encode multiple distinct ABC importers to facilitate the acquisition of 51 essential nutrients such as sugars, amino acids, vitamins, compatible solutes, and metal ions 1, 7 . Many 52 ABC importers can transport more than one substrate using high-affinity interactions between SBPs 53 and transported ligands (herein termed cognate substrates) 2 . Despite low sequence similarity between 54 SBPs of different ABC importers, they share a common architecture comprising two structurally 55 conserved rigid lobes connected by a flexible hinge region (Figure 1) 2 . Numerous biophysical 8 and 56 structural analyses 9 indicate that ligand binding at the interface of the two lobes facilitates switching 57 between two conformations, i.e. from an open to a closed conformation. Bending and unbending of the 58 hinge region brings the two lobes together (closed conformation) or apart (open conformation), 59 respec...

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