title: Watching conformational dynamics of ABC transporters with single-molecule tools.
abstract: 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.
authors.affiliation: Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Florence
authors.initials: F
authors.lastname: Husada
authors.affiliation: Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, Netherlands Proteomics Centre & Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Giorgos
authors.initials: G
authors.lastname: Gouridis
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, Netherlands Proteomics Centre & Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Ruslan
authors.initials: R
authors.lastname: Vietrov
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, Netherlands Proteomics Centre & Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Gea K
authors.initials: GK
authors.lastname: Schuurman-Wolters
authors.affiliation: Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Ploetz
authors.affiliation: Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Marijn
authors.initials: M
authors.lastname: de Boer
authors.affiliation: Department of Biochemistry, Groningen Biomolecular Science and Biotechnology Institute, Netherlands Proteomics Centre & Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands b.poolman@rug.nl t.m.cordes@rug.nl.
authors.forename: Bert
authors.initials: B
authors.lastname: Poolman
authors.affiliation: Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands b.poolman@rug.nl t.m.cordes@rug.nl.
authors.forename: Thorben
authors.initials: T
authors.lastname: Cordes
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002232:Carbocyanines / Q000737:chemistry; D002850:Chromatography, Gel / Q000379:methods; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D015203:Reproducibility of Results
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002232:Carbocyanines; C089187:cyanine dye 3; C085321:cyanine dye 5
keywords: ABC importer; FRET; fluorophore labelling; single-molecule studies; transport mechanism