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.
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October 2015
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Review Article|
October 09 2015
Watching conformational dynamics of ABC transporters with single-molecule tools
Florence Husada;
Florence Husada
*Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
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Giorgos Gouridis;
Giorgos Gouridis
*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
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Ruslan Vietrov;
Ruslan Vietrov
†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
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Gea K. Schuurman-Wolters;
Gea K. Schuurman-Wolters
†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
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Evelyn Ploetz;
Evelyn Ploetz
*Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
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Marijn de Boer;
Marijn de Boer
*Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
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Bert Poolman;
Bert Poolman
1
†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
1Correspondence may be addressed to either author (emailb.poolman@rug.nl or t.m.cordes@rug.nl).
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Thorben Cordes
Thorben Cordes
1
*Molecular Microscopy Research Group & Single-molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
1Correspondence may be addressed to either author (emailb.poolman@rug.nl or t.m.cordes@rug.nl).
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Publisher: Portland Press Ltd
Received:
June 05 2015
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© 2015 Authors; published by Portland Press Limited
2015
Biochem Soc Trans (2015) 43 (5): 1041–1047.
Article history
Received:
June 05 2015
Citation
Florence Husada, Giorgos Gouridis, Ruslan Vietrov, Gea K. Schuurman-Wolters, Evelyn Ploetz, Marijn de Boer, Bert Poolman, Thorben Cordes; Watching conformational dynamics of ABC transporters with single-molecule tools. Biochem Soc Trans 1 October 2015; 43 (5): 1041–1047. doi: https://doi.org/10.1042/BST20150140
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