Protein induced fluorescence enhancement (PIFE) for probing protein–nucleic acid interactions

Hwang, Helen; Myong, Sua

doi:10.1039/c3cs60201j

Cited by 213 publications

(212 citation statements)

References 25 publications

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“…Cy3 fluorescence is enhanced when in the vicinity of a protein as first noted in studies of UvrD[24] and this has been used to study the binding of many proteins to nucleic acids[25–36]. This is also the case for hRPA (Figure 4).…”

Section: Resultsmentioning

confidence: 69%

“…We also designed a novel set of chimeric DNA molecules to calibrate the dependence of the Cy3 fluorescence enhancement on the distance (along the contour length) between the bound hRPA and the Cy3 fluorophore on 3′-Cy3-(dT) L . The origin of the protein-induced Cy3 fluorescence enhancement has been proposed to be due to an effect on the photo-induced cis-trans isomerization within the Cy3 fluorophore[34, 36, 42]. The ssDNA molecules used in our study are very flexible, hence transient loop formation can bring the Cy3 in direct contact with the hRPA.…”

Section: Discussionmentioning

confidence: 99%

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Diffusion of Human Replication Protein A along Single-Stranded DNA

Nguyen

Sokoloski

Galletto

et al. 2014

Journal of Molecular Biology

135

245

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Replication Protein A (RPA) is a eukaryotic single stranded (ss) DNA binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA-DNA interactions, we combined ensemble and single molecule fluorescence approaches to examine human RPA diffusion along ssDNA and find that an hRPA hetero-trimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D1 ~5000 nucleotide2s−1 at 37°C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.

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Section: Resultsmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Diffusion of Human Replication Protein A along Single-Stranded DNA

Nguyen

Sokoloski

Galletto

et al. 2014

Journal of Molecular Biology

135

245

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“…Cy3 exhibits an enhanced fluorescence quantum yield when it is located in a protein environment, a phenomenon referred to as PIFE (22,23). 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).…”

Section: Discussionmentioning

confidence: 99%

“…The static receptor population was excluded from further analysis. The state of higher fluorescence intensity in the fluctuating trajectories likely arises from protein-induced fluorescence enhancement (PIFE) of Cy3 (22,23). Accordingly, for each single molecule, we normalized the fluorescence intensities at each time point to the mean intensity of the lower intensity state (Fig.…”

Section: Resultsmentioning

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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“…ScPif1 is an SF1B 5′ to 3′ ssDNA translocase that translocates with a rate of 81 ± 8 nt/s on a poly(dT) track at saturating [ATP] [50 mM Tris·HCl, pH 8.3, 100 mM NaCl, 5 mM MgCl 2 , 0.5 mM DTT, and 20% (wt/vol) glycerol, 22°C] (28). The arrival of Pif1 at the 3′ end of the 3′-dT-Cy3-(dT) 140 results in an enhancement of Cy3 fluorescence intensity that can be used to monitor ssDNA translocation (25,28,49) and has been referred to as PIFE (protein-induced fluorescence enhancement) (50,51). Pif1 also binds preferentially to an ss/double-strand (ds)DNA junction and remains bound to the junction while undergoing ATP-dependent ssDNA translocation, resulting in the transient formation of an ssDNA loop (41).…”

mentioning

confidence: 99%

Chemo-mechanical pushing of proteins along single-stranded DNA

Sokoloski

Козлов

Galletto

et al. 2016

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

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Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3′-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5′ to 3′ ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5′ to 3′) pushing of the SSB toward the 3′ ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3′ to 5′) direction, are observed with EcRep and EcUvrD (both 3′ to 5′ ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.SSB proteins | SF1 translocases | DNA motors | dynamics

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Protein induced fluorescence enhancement (PIFE) for probing protein–nucleic acid interactions

Cited by 213 publications

References 25 publications

Diffusion of Human Replication Protein A along Single-Stranded DNA

Diffusion of Human Replication Protein A along Single-Stranded DNA

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

Chemo-mechanical pushing of proteins along single-stranded DNA

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Protein induced fluorescence enhancement (PIFE) for probing protein–nucleic acid interactions

Cited by 213 publications

References 25 publications

Diffusion of Human Replication Protein A along Single-Stranded DNA

Diffusion of Human Replication Protein A along Single-Stranded DNA

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

Chemo-mechanical pushing of proteins along single-stranded DNA

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