Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments

Jazi, Atieh Aminian; Ploetz, Evelyn; Arizki, Muhamad; Balasubramaniam, Dhandayuthapani; Waclawska, Izabela; Krämer, Reinhard; Ziegler, Christine; Cordes, Thorben

doi:10.1021/acs.biochem.6b00916

Cited by 15 publications

(24 citation statements)

References 60 publications

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“…Over the past years [13], various labs have introduced single-molecule tools to investigate the structural dynamics of active membrane transporters [14][15][16][17][18][19][20][21][22][23][24][25]. Förster resonance energy transfer in combination with single-molecule detection (smFRET [26][27][28]) has proven to be a particularly useful tool for the validation of structural models [29][30][31], and for revealing functional features of transporters which are mechanistically important, such as conformational heterogeneity [28,[32][33][34].…”

Section: Introductionmentioning

confidence: 99%

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

Tassis

Vietrov

Koning

et al. 2020

Preprint

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The current model of active transport via ABC importers is mostly based on structural, biochemical and genetic data. We here establish single-molecule Förster-resonance energy transfer (smFRET) assays to monitor the conformational states and heterogeneity of the type-I ABC importer OpuA from Lactococcus lactis. Our studies include intradomain assays that elucidate conformational changes within the substrate-binding domain (SBD) OpuAC and interdomain assays between SBDs or transmembrane domains. Using the methodology, we studied ligand-binding mechanisms as well as ATP and glycine betaine dependences of conformational changes. Our study expands the scope of smFRET investigations towards a class of so far unstudied ABC importers, and paves the way for a full understanding of their transport cycle in the future.

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Section: Introductionmentioning

confidence: 99%

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

Tassis

Vietrov

Koning

et al. 2020

Preprint

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“…By introducing a single cysteine per domain and stochastic labeling, hence two cysteine residues per complex in case of a homodimeric complex, it will be possible to observe interdomain movements (Figure 5b), as has been shown for the ABC-transporter BtuCD by labelling the transmembrane domains 23 , the ABC-transporter MRP1 by labelling the NBD's 24 , but also for the ABC-transporters MsbA 6 and McjD 7 . A similar approach has been used in smFRET studies on BetP, a homotrimeric protein with three fluorophores per complex 8 . Alternatively, one could label the protein with a fluorescence donor and introduce a fluorescence quencher in the ligand or membrane to probe conformational dynamics.…”

Section: Discussionmentioning

confidence: 99%

“…Even though all these techniques can give information on protein dynamics, for instance using caged compounds and free-electron lasers in serial crystallography 3 , or imaging under turnover conditions in CryoEM 4 , transient states and continuous dynamics are not readily obtained. Double electron-electron resonance (DEER) or Pulsed Electronelectron double resonance (PELDOR) can be used to probe distance changes upon changing conditions 5 and single-molecule Förster Resonance Energy Transfer (smFRET) can be used to obtain single molecule dynamics of proteins and other macromolecular assemblies [6][7][8][9][10] . The latter two techniques make use of two labels that are introduced for instance by attaching them to cysteine residues via maleimide 11 or methanethiosulfonate chemistry 12 or introducing them as non-natural amino acids 13 .…”

Section: Introductionmentioning

confidence: 99%

In Silico Method for Selecting Residue Pairs for Single-Molecule Microscopy and Spectroscopy

Sikkema

Poolman

2020

Preprint

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Obtaining (dynamic) structure related information on proteins is key for understanding their function. Methods as single-molecule Förster Resonance Energy Transfer (smFRET) and Electron Paramagnetic Resonance (EPR) that measure distances between labeled residues to obtain dynamic information rely on selection of suitable residue pairs for chemical modification. Selection of pairs of amino acids, that show sufficient distance changes upon activity of the protein, can be a tedious process. Here we present an in silico approach that makes use of two or more structures (or structure models) to filter suitable residue pairs for FRET or EPR from all possible pairs within the protein. We apply the method for the study of the conformational dynamics of the substrate-binding domain of the osmoregulatory ATP-Binding Cassette transporter OpuA. This method speeds up the process of designing mutants, and because of its systematic nature, the chances of missing promising candidates are reduced.

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“…Confocal scanning experiments were performed at room temperature and using a home-built confocal scanning microscope as described previously 29, 68, 69 . In brief, surface scanning was performed using a XYZ-piezo stage with 100×100×20 µm range (P-517-3CD with E-725.3CDA, Physik Instrumente).…”

Section: Methodsmentioning

confidence: 99%

Conformational and dynamical plasticity in substrate-binding proteins underlies selective transport in ABC importers

Boer

Gouridis

Vietrov

et al. 2019

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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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Caging and Photoactivation in Single-Molecule Förster Resonance Energy Transfer Experiments

Cited by 15 publications

References 60 publications

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

Single-molecule studies of conformational states and dynamics in the ABC importer OpuA

In Silico Method for Selecting Residue Pairs for Single-Molecule Microscopy and Spectroscopy

Conformational and dynamical plasticity in substrate-binding proteins underlies selective transport in ABC importers

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