Watching conformational dynamics of ABC transporters with single-molecule tools

Husada, Florence; Gouridis, Giorgos; Vietrov, Ruslan; Schuurman-Wolters, Gea K.; Ploetz, Evelyn; Boer, Marijn de; Poolman, Bert; Cordes, Thorben

doi:10.1042/bst20150140

Cited by 35 publications

(44 citation statements)

References 34 publications

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“…The effect of the D723R mutation also dramatically demonstrates the long range allosteric relay of the conformational change (Pines et al, 2011). Indeed, the switching time is very long compared to other protein conformational transitions (Hammes, 2002; Husada et al, 2015), indicating a complex energy profile with multiple steps.…”

Section: Discussionmentioning

confidence: 99%

Force regulated conformational change of integrin αVβ3

et al. 2017

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Integrins mediate cell adhesion to extracellular matrix and transduce signals bidirectionally across the membrane. Integrin αVβ3 has been shown to play an essential role in tumor metastasis, angiogenesis, hemostasis and phagocytosis. Integrins can take several conformations, including the bent and extended conformations of the ectodomain, which regulate integrin functions. Using a biomembrane force probe, we characterized the bending and unbending conformational changes of a single integrin αVβ3 molecule on a living cell surface in real-time. We measured the probabilities of conformational changes, rates and speeds of conformational transitions, and the dynamic equilibrium between the two conformations, which were regulated by tensile force, dependent on the ligand, and altered by point mutations. These findings provide insights into how αVβ3 acts as a molecular machine and how its physiological function and molecular structure are coupled at the single molecule level.

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Section: Discussionmentioning

confidence: 99%

Force regulated conformational change of integrin αVβ3

et al. 2017

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“…Unfortunately, no established quality criteria exist. Optimized labeling protocols 25 and using a bias-free diffusion-based method can prevent “cherry picking” when individual immobilized molecules are being studied at later stages.…”

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confidence: 99%

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

et al. 2017

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Caged organic fluorophores are established tools for localization-based super-resolution imaging. Their use relies on reversible deactivation of standard organic fluorophores by chemical reduction or commercially available caged dyes with ON switching of the fluorescent signal by ultraviolet (UV) light. Here, we establish caging of cyanine fluorophores and caged rhodamine dyes, i.e., chemical deactivation of fluorescence, for single-molecule Förster resonance energy transfer (smFRET) experiments with freely diffusing molecules. They allow temporal separation and sorting of multiple intramolecular donor–acceptor pairs during solution-based smFRET. We use this “caged FRET” methodology for the study of complex biochemical species such as multisubunit proteins or nucleic acids containing more than two fluorescent labels. Proof-of-principle experiments and a characterization of the uncaging process in the confocal volume are presented. These reveal that chemical caging and UV reactivation allow temporal uncoupling of convoluted fluorescence signals from, e.g., multiple spectrally similar donor or acceptor molecules on nucleic acids. We also use caging without UV reactivation to remove unwanted overlabeled species in experiments with the homotrimeric membrane transporter BetP. We finally outline further possible applications of the caged FRET methodology, such as the study of weak biochemical interactions, which are otherwise impossible with diffusion-based smFRET techniques because of the required low concentrations of fluorescently labeled biomolecules.

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“…Liganded crystals not only identified unexpected substrates but also revealed how the specificity can be changed dramatically by a single alteration in a residue distant to the binding site. Thorben Cordes followed with a fascinating presentation of mechanistic detail derived from single-molecule FRET studies of SBPs, challenging the classical ‘Venus Flytrap’ model [ 49 , 50 ].…”

Section: Structure and Mechanism: An Ideal Pas De Deux mentioning

confidence: 99%

ABC transporter research: going strong 40 years on

Theodoulou

Kerr

2015

Biochemical Society Transactions

259

150

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In most organisms, ABC transporters constitute one of the largest families of membrane proteins. In humans, their functions are diverse and underpin numerous key physiological processes, as well as being causative factors in a number of clinically relevant pathologies. Advances in our understanding of these diseases have come about through combinations of genetic and protein biochemical investigations of these transporters and the power of in vitro and in vivo investigations is helping to develop genotype–phenotype understanding. However, the importance of ABC transporter research goes far beyond human biology; microbial ABC transporters are of great interest in terms of understanding virulence and drug resistance and industrial biotechnology researchers are exploring the potential of prokaryotic ABC exporters to increase the capacity of synthetic biology systems. Plant ABC transporters play important roles in transport of hormones, xenobiotics, metals and secondary metabolites, pathogen responses and numerous aspects of development, all of which are important in the global food security area. For 3 days in Chester, this Biochemical Society Focused Meeting brought together researchers with diverse experimental approaches and with different fundamental questions, all of which are linked by the commonality of ABC transporters.

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Watching conformational dynamics of ABC transporters with single-molecule tools

Cited by 35 publications

References 34 publications

Force regulated conformational change of integrin αVβ3

Force regulated conformational change of integrin αVβ3

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

ABC transporter research: going strong 40 years on

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