Demystifying PIFE: The Photophysics Behind the Protein-Induced Fluorescence Enhancement Phenomenon in Cy3

Stennett, Elana M. S.; Ciuba, Monika A.; Lin, Su; Levitus, Marcia

doi:10.1021/acs.jpclett.5b00613

Cited by 109 publications

(130 citation statements)

References 41 publications

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“…The mechanism of fluorescence reduction due to RFS-1/RIP-1 binding to the 5′ end of the filament may represent a modulation of the efficiency of RAD-51-induced PIFE, such that the overall equilibrium PIFE of 5′ Cy3-labeled ssDNA for the RAD-51/RFS-1/RIP-1 complex is lower than that for RAD-51 alone. Recent evidence suggests that Cy3 PIFE arises due to decreased photoisomerization of the dye to the non-fluorescent cis when sterically constrained by protein (Stennett et al., 2015). Indeed, our single molecule fluorescence resonance energy transfer (FRET) studies also established that RFS-1/RIP-1 converts RAD-51-ssDNA filaments to a more flexible conformation (Taylor et al., 2015).…”

Section: Resultsmentioning

confidence: 99%

A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling

et al. 2016

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SummaryCentral to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5′ end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5′→3′ polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs.

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Section: Resultsmentioning

confidence: 99%

A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling

et al. 2016

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“…Recently, it was shown that PIFE is specifically due to a reduction in excited-state cis-trans isomerization of Cy3, which normally competes with fluorescence emission (26). Hence, the emission intensity of Cy3 is determined by the degree of steric restriction imposed on the fluorophore by the local environment: Highly restricted environments that inhibit cis-trans isomerization will produce relatively bright emission, whereas unrestricted environments that permit isomerization will lead to relatively weak emission.…”

Section: Discussionmentioning

confidence: 99%

Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β ₂ AR

Lamichhane

Liu

Pljevaljčić

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

120

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Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCRtargeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human β 2 -adrenergic receptor (β 2 AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.signal transduction mechanisms | agonists and inverse agonists | conformational polymorphism | single-molecule fluorescence spectroscopy | phospholipid nanodiscs G protein-coupled receptors (GPCRs) mediate a multitude of physiological functions and are the targets for a myriad of drugs (1), many of which elicit different functional outcomes through the same receptor (2). It remains to be rationalized at the molecular level why some drugs stimulate the signaling activity of a GPCR (full or partial agonists), whereas others either repress the receptor (inverse agonists) or have no effect on the intrinsic signaling activity (neutral antagonists). Moreover, the existence of a high basal activity of some GPCRs (3) suggests that the conformational transitions leading to activation may occur spontaneously, even in the absence of ligands, which in turn raises questions about the mechanistic roles of GPCR ligands. Understanding the mechanisms and pathways of receptor activation or deactivation, and how these are linked to the binding of ligands with different chemical structures and pharmacological efficacies, will aid in design of new GPCR-targeted drugs with tailored pharmacological responses and fewer side effects. To attain these goals, new methods are required to visualize the conformational dynamics of GPCRs in the presence and absence of drugs.The β 2 -adrenergic receptor (β 2 AR) has been extensively investigated in crystals (4, 5), by NMR in solution (6-11), by bulk fluorescence spectroscopy in s...

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“…For the experiments, we took advantage of an inherent property of the Cyanine 3 fluorophore (Cy3). When Cy3 is covalently attached to DNA it displays a higher fluorescence intensity that depends on the proximity of the fluorophore to a nearby bound protein, a phenomenon termed protein-induced fluorescence enhancement (PIFE) 9,10 (Fig. 1b).…”

Section: Introductionmentioning

confidence: 99%

An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases

Gunn

Marko

Mondragón

2017

Nat Struct Mol Biol

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Topoisomerases are enzymes involved in maintaining the topological state of cellular DNA. Despite many structural, biophysical, and biochemical studies, their dynamic characteristics remain poorly understood. Recent single molecule experiments revealed that an important feature of the type IA topoisomerase mechanism is the presence of pauses between relaxation events. However, these experiments cannot determine whether the protein remains DNA bound during the pauses or the relationship between domain movements in the protein and topological changes in the DNA. By combining two orthogonal single molecule techniques, we observed that topoisomerase IA is constantly changing conformation and attempting to modify the topology of DNA, but only succeeds in a fraction of the attempts. Thus, its mechanism can be described as a series of DNA strand passage attempts that culminate in a successful relaxation event.

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Demystifying PIFE: The Photophysics Behind the Protein-Induced Fluorescence Enhancement Phenomenon in Cy3

Cited by 109 publications

References 41 publications

A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling

A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling

Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β ₂ AR

An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases

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Demystifying PIFE: The Photophysics Behind the Protein-Induced Fluorescence Enhancement Phenomenon in Cy3

Cited by 109 publications

References 41 publications

A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling

A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling

Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β 2 AR

An orthogonal single-molecule experiment reveals multiple-attempt dynamics of type IA topoisomerases

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Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β ₂ AR