Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

Ploetz, Evelyn; Lerner, Eitan; Husada, Florence; Roelfs, Martin; Chung, SangYoon; Hohlbein, Johannes; Weiss, Shimon; Cordes, Thorben

doi:10.1038/srep33257

Cited by 80 publications

(113 citation statements)

References 121 publications

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“…Neither the introduction of cysteines nor the addition of fluorescent labels affected the activity of the transporter in proteoliposomes; this is indicated by similar basal and ligand‐induced ATPase activity compared to wild‐type McjD (Fig 1D). All interpretations given below are supported by comparison of the observed E* values of McjD with respect to static dsDNA ruler structures (Fig EV1A and B) that we have previously described (Ploetz et al , 2016). …”

Section: Resultssupporting

confidence: 82%

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

et al. 2018

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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.

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Section: Resultssupporting

confidence: 82%

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

et al. 2018

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“…As a final comment, we note that our data analysis approach to derive the distance ratio of two (conformational) states with altered quantum yield of donor/acceptor dye could also be applied for situations where FRET is changed due to protein-induced fluorescence enhancement (PIFE) 41–43 . The approach suggested here is particularly attractive for PIFE, since the distance ratio is independent of the donor quantum yield, and thus Cy3, which is the most popular dye for PIFE, could be used in a straightforward fashion without additional knowledge of its quantum yield (changes).…”

Section: Discussionmentioning

confidence: 99%

Single-molecule observation of ligand binding and conformational changes in FeuA

Boer

Gouridis

Muthahari

et al. 2019

Preprint

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14The specific binding of ligands by proteins and the coupling of this process to conformational changes 15 are fundamental to protein function. We designed a fluorescence-based single-molecule assay and data 16 analysis procedure that allows the simultaneous real-time observation of ligand binding and 17 conformational changes in FeuA. The substrate-binding protein FeuA binds the ligand ferri-18 bacillibactin and delivers it to the ABC importer FeuBC, which is involved in iron uptake in bacteria. 19The conformational dynamics of FeuA was assessed via Förster resonance energy transfer (FRET), 20 whereas the presence of the ligand was probed by fluorophore quenching. We reveal that ligand 21 binding shifts the conformational equilibrium of FeuA from an open to a closed conformation. Ligand 22 binding occurs via an induced-fit mechanism, i.e., the ligand binds to the open state and subsequently 23 triggers a rapid closing of the protein. However, FeuA also rarely samples the closed conformation 24 without the involvement of the ligand. This shows that ligand interactions are not required for 25 conformational changes in FeuA. However, ligand interactions accelerate the conformational change 26 10000-fold and temporally stabilize the formed conformation 250-fold. 27 28 29 2 SIGNIFICANCE STATEMENT 30Ligand binding and the coupling of this process to conformational changes in proteins are 31 fundamental to their function. We developed a single-molecule approach that allows the 32 simultaneous observation of ligand binding and conformational changes in the substrate-33 binding protein FeuA. This allows to directly observe the ligand binding process, ligand-34 driven conformational changes as well as rare short-lived conformational transitions that are 35 uncoupled from the ligand. These findings provide insight into the fundamental relation 36 between ligand-protein interactions and conformational changes. Our findings are, however, 37 not only of interest to understand protein function, but the developed data analysis procedure 38 allows the determination of (relative) distance changes in single-molecule FRET experiments, 39for situations in which donor and acceptor fluorophore are influenced by quenching processes. 40 41 42 48 the energy landscape that connect unliganded and liganded conformational states (Fig. 1). In the 49 induced-fit mechanism, ligand interactions trigger conformational changes, whereas in the 50 conformational selection mechanism, ligand interactions selectively stabilize a subset of 51 conformations that are already present in the unliganded protein ( Fig. 1). Both mechanisms require 52 intermediate states that are formed during the ligand-binding process. For example, when a protein 53 switches between two conformational states, such as an open and closed conformation ( Fig. 1), an 54 open-liganded state in the induced-fit mechanism or a closed-unliganded state in the conformational 55 3 selection mechanism, are essential intermediate states. However, the study of such transient and 56 thermody...

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“…PIFE-FRET thus provided direct evidence for molecular coordination in the open transcription bubble (Fig. 6C) (104). Similarly, smFRET was combined with photo-induced electron transfer (PET) (105), where the donor dye was quenched by a nearby tryptophan moiety.…”

Section: Toward Multiple Reaction Coordinatesmentioning

confidence: 93%

“…ALEX can therefore distinguish molecules with different numbers of donor and acceptor dyes, or molecules with altered dye fluorescence quantum yields. In a recent implementation of ALEX to simultaneously report two distances, smFRET was combined with protein-induced fluorescence enhancement (PIFE) (103,104). One distance was between the dyes and within the FRET distance range (~3 to 9 nm); the other, between one dye and a bound protein, was in the shorter PIFE distance range (<3 nm).…”

Section: Toward Multiple Reaction Coordinatesmentioning

confidence: 99%

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Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

Lerner

Cordes

Ingargiola

et al. 2018

Science

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458

449

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Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.

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Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers

Cited by 80 publications

References 121 publications

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

Single-molecule observation of ligand binding and conformational changes in FeuA

Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

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