Fluorescence Resonance Energy Transfer Pair Research Articles

G-Tetraplex-Induced FRET within Telomeric Repeat Sequences Using (Py) A-(Per) A as Energy Donor-Acceptor Pair.

G-tetraplex induced fluorescence resonance energy transfer (FRET) within telomeric repeat sequences has been studied using a nucleoside-tethered FRET pair embedded in the human telomeric G-quadruplex forming sequence (5'-A GGG TT(Py) A GGG TT(Per) A GGG TTA GGG-3', Py=pyrene, Per=perylene). Conformational change from a single strand to an anti-parallel G-quadruplex leads to FRET from energy donor ((Py) A) to acceptor ((Per) A). The distance between the FRET donor/acceptor partners was controlled by changing the number of G-quartet spacer units. The FRET efficiency decreases with increase in G-quartet units. Overall findings indicate that this could be further used for the development of FRET-based sensing and measurement techniques.

Fluorescent Protein Based FRET Pairs with Improved Dynamic Range for Fluorescence Lifetime Measurements

Fluorescence Resonance Energy Transfer (FRET) using fluorescent protein variants is widely used to study biochemical processes in living cells. FRET detection by fluorescence lifetime measurements is the most direct and robust method to measure FRET. The traditional cyan-yellow fluorescent protein based FRET pairs are getting replaced by green-red fluorescent protein variants. The green-red pair enables excitation at a longer wavelength which reduces cellular autofluorescence and phototoxicity while monitoring FRET. Despite the advances in FRET based sensors, the low FRET efficiency and dynamic range still complicates their use in cell biology and high throughput screening. In this paper, we utilized the higher lifetime of NowGFP and screened red fluorescent protein variants to develop FRET pairs with high dynamic range and FRET efficiency. The FRET variations were analyzed by proteolytic activity and detected by steady-state and time-resolved measurements. Based on the results, NowGFP-tdTomato and NowGFP-mRuby2 have shown high potentials as FRET pairs with large fluorescence lifetime dynamic range. The in vitro measurements revealed that the NowGFP-tdTomato has the highest Förster radius for any fluorescent protein based FRET pairs yet used in biological studies. The developed FRET pairs will be useful for designing FRET based sensors and studies employing Fluorescence Lifetime Imaging Microscopy (FLIM).

Novel fluorescence resonance energy transfer-based reporter reveals differential calcineurin activation in neonatal and adult cardiomyocytes.

Novel fluorescence resonance energy transfer-based genetically encoded reporters of calcineurin are constructed by fusing the two subunits of calcineurin with P2A-based linkers retaining the expected native conformation of calcineurin. Calcineurin reporters display robust responses to calcium transients in HEK293 cells. The sensor responses are correlated with NFATc1 translocation dynamics in HEK293 cells. The sensors are uniformly distributed in neonatal myocytes and respond efficiently to single electrically evoked calcium transients and show cumulative activation at frequencies of 0.5 and 1 Hz. In adult myocytes, the calcineurin sensors appear to be localized to the cardiac z-lines, and respond to cumulative calcium transients at frequencies of 0.5 and 1 Hz. The phosphatase calcineurin is a central component of many calcium signalling pathways, relaying calcium signals from the plasma membrane to the nucleus. It has critical functions in a multitude of systems, including immune, cardiac and neuronal. Given the widespread importance of calcineurin in both normal and pathological conditions, new tools that elucidate the spatiotemporal dynamics of calcineurin activity would be invaluable. Here we develop two separate genetically encoded fluorescence resonance energy transfer (FRET)-based sensors of calcineurin activation, DuoCaN and UniCaN. Both sensors showcase a large dynamic range and rapid response kinetics, differing primarily in the linker structure between the FRET pairs. Both sensors were calibrated in HEK293 cells and their responses correlated well with NFAT translocation to the nucleus, validating the biological relevance of the sensor readout. The sensors were subsequently expressed in neonatal rat ventricular myocytes and acutely isolated adult guinea pig ventricular myocytes. Both sensors demonstrated robust responses in myocytes and revealed kinetic differences in calcineurin activation during changes in pacing rate for neonatal versus adult myocytes. Finally, mathematical modelling combined with quantitative FRET measurements provided novel insights into the kinetics and integration of calcineurin activation in response to myocyte Ca transients. In all, DuoCaN and UniCaN stand as valuable new tools for understanding the role of calcineurin in normal and pathological signalling.

High Affinity Host-Guest FRET Pair for Single-Vesicle Content-Mixing Assay: Observation of Flickering Fusion Events.

Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.

Close spatio-association of the transient receptor potential canonical 4 (TRPC4) channel with Gαi in TRPC4 activation process.

TPRC channels are Ca(2+)-permeable, nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). TRPC4 is commonly assumed to be activated by Gq/phospholipase C-coupled receptors. However, the other molecular mechanisms by which Gα proteins regulate TRPC4 remain unclear. Here, we found that Gαi2 regulates TRPC4 activation by direct binding. To investigate this mechanism, we used whole patch clamp and fluorescence resonance energy transfer (FRET). We tagged an isoform of mTRPC4 and G protein with CFP and YFP, respectively, and transiently transfected cells with the FRET pair. The FRET efficiency between TRPC4β-CFP and the constitutively active mutant form of Gαi2 was nearly 15% and was greater than that observed with wild-type Gαi2 (nearly 5%). Gβγ and the TRPC4 channel showed a FRET efficiency lower than 6%. In HEK293 cells transfected with the M2 muscarinic receptor, the application of carbachol increased the FRET efficiency between TRPC4β-CFP and Gαi2(WT)-YFP from 4.7 ± 0.4% (n = 7) to 12.6 ± 1.4% (n = 7). We also found that the TRPC4 channel directly interacts with Gαi2, but not with Gαq, when the channel is open. We analyzed the calcium levels in HEK293 cells expressing the channels and Gαi2 or Gαq using the calcium indicator YC6.1 (Yellow Cameleon 6.1). In response to the muscarinic agonist carbachol, M2-, Gαi2-, and TRPC4-expressing cells showed a prolonged Ca(2+) influx compared with cells expressing only M2. Together, these data suggest that Gαi2 activates the TRPC4 channel by direct binding, which then induces Ca(2+) entry.

Nanoscale energy-route selector consisting of multiple photo-switchable fluorescence-resonance-energy-transfer structures on DNA

We report on a nanoscale energy-route selector consisting of multiple fluorescence resonance energy transfer (FRET) structures switched by external signaling with multiple wavelengths of light. In each FRET structure, a specific activator molecule is incorporated to a FRET pair of a donor and an acceptor to control the activation of the acceptor. Owing to this configuration, the FRET structures are switched independently, and an energy route is selected. Two photo-switchable FRET structures, one consists of Alexa Fluor 568 (donor), Cy5 (acceptor), and Alexa Fluor 405 (activator), and the other consists of Alexa Fluor 568 (donor), Cy5.5 (acceptor), and Cy3 (activator), were constructed using DNA strands modified with fluorescence molecules. Switching rates for the individual FRET structures were measured as 64 and 49 %, respectively. An energy-route selector was then assembled with the FRET structures which share a single donor. Experimental results demonstrate that the energy route can be changed repeatedly by activation control using three wavelengths of light.

Dual-Switching FRET (dsFRET) Imaging Based on Photoswitchable Donor-Acceptor Pair

Fluorescence resonance energy transfer (FRET), especially with fluorescent proteins of donor and acceptor, has been widely used to measure biomolecular interactions. To overcome limitations of existing approaches for quantitative FRET, we here put forward a novel platform of dual-switching FRET (dsFRET), with a photoswitchable donor as well as a photoswitchable acceptor. With the photoswitchable capability from the FRET pair we constructed, neither donor-only nor acceptor-only samples would be required as control reference for calculation. Experiments of dsFRET and traditional 33-FRET were conducted and compared in both dimer and two-hybrid forms. Our data demonstrate that dsFRET has higher accuracy and stability than 33 -FRET, mainly benefited from in-situ references. Further development of dsFRET has been pursued, to enhance the performance of this new methodology, and also to extend its applications, such as in subcellular FRET and in-vivo FRET.

Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes.

Pairs of membrane-associated molecules exhibiting fluorescence resonance energy transfer (FRET) provide a sensitive technique to measure changes in a cell's membrane potential. One of the FRET pair binds to one surface of the membrane and the other is a mobile ion that dissolves in the lipid bilayer. The voltage-related signal can be measured as a change in the fluorescence of either the donor or acceptor molecules, but measuring their ratio provides the largest and most noise-free signal. This technology has been used in a variety of ways; three are documented in this chapter: (1) high throughput drug screening, (2) monitoring the activity of many neurons simultaneously during a behavior, and (3) finding synaptic targets of a stimulated neuron. In addition, we provide protocols for using the dyes on both cultured neurons and leech ganglia. We also give an updated description of the mathematical basis for measuring the coherence between electrical and optical signals. Future improvements of this technique include faster and more sensitive dyes that bleach more slowly, and the expression of one of the FRET pair genetically.

FRET Imaging by Laser Scanning Cytometry on Large Populations of Adherent Cells.

The application of FRET (fluorescence resonance energy transfer) sensors for monitoring protein-protein interactions under vital conditions is attracting increasing attention in molecular and cell biology. Laser-scanning cytometry (LSC), a slide-based sister procedure to flow cytometry, provides an opportunity to analyze large populations of adherent cells or 2-D solid tissues in their undisturbed physiological settings. Here we provide an LSC-based three-laser protocol for high-throughput ratiometric FRET measurements utilizing cyan and yellow fluorescent proteins as a FRET pair. Membrane labeling with Cy5 dye is used for cell identification and contouring. Pixel-by-pixel and single-cell FRET efficiencies are calculated to estimate the extent of the molecular interactions and their distribution in the cell populations examined. We also present a non-high-throughput donor photobleaching FRET application, for obtaining the required instrument parameters for ratiometric FRET. In the biological model presented, HeLa cells are transfected with the ECFP- or EYFP-tagged Fos and Jun nuclear proteins, which heterodimerize to form active AP1 transcription factor.

Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain.

Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviors. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs) has the potential to meet this challenge. Currently prevalent GEVIs are based on the voltage-sensitive fluorescent protein (VSFP) prototypical design or on the voltage-dependent state transitions of microbial opsins. We recently introduced a new VSFP design in which the voltage-sensing domain (VSD) is sandwiched between a fluorescence resonance energy transfer pair of fluorescent proteins (termed VSFP-Butterflies) and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians.

Tumor-marker-mediated "on-demand" drug release and real-time monitoring system based on multifunctional mesoporous silica nanoparticles.

"On-demand" drug release can maximize therapeutic efficacy for specific states of malignancies and minimize drug toxicity to healthy cells. Meanwhile, there is lack of a real-time monitoring platform to accurately investigate the amount of anticancer drugs released, especially nonfluorescent ones. So it is significant to integrate both issues in one ideal drug delivery system. To achieve this, here we present a novel stimuli-responsive controlled drug delivery system toward the tumor marker survivin mRNA, using a real-time monitoring approach based on the fluorescence resonance energy transfer (FRET) strategy to quantify the process of drug release. First, 7-amino-4-methlcoumarin (AMCA) dye terminated short oligonucleotide (FlareA) will hybridize with fluorescein isothiocyanate (FITC) labeled long oligonucleotide (S1F), which contains a recognition element to a specific RNA transcript, to form a FRET pair capped on the pores of mesoporous silica nanoparticles (MSNs). Following a target-recognition reaction, the target with a longer strand displaces the FlareA strand to form a longer and more stable duplex with S1F, which leads to the removal of the capped oligonucleotide from the MSNs and triggers the release of the entrapped cargo while FRET between AMCA and FITC is broken. The relevant change in donor and acceptor fluorescence signal can be used to monitor the unlocking and release event in real-time. Further investigations have also demonstrated that this release system possesses the capacity of modulating the extent of drug release according to the cell states, giving the platform an equally broad spectrum of applications in anticancer therapy.

FRET based quantification and screening technology platform for the interactions of leukocyte function-associated antigen-1 (LFA-1) with intercellular adhesion molecule-1 (ICAM-1).

The interaction between leukocyte function-associated antigen-1(LFA-1) and intercellular adhesion molecule-1 (ICAM-1) plays a pivotal role in cellular adhesion including the extravasation and inflammatory response of leukocytes, and also in the formation of immunological synapse. However, irregular expressions of LFA-1 or ICAM-1 or both may lead to autoimmune diseases, metastasis cancer, etc. Thus, the LFA-1/ICAM-1 interaction may serve as a potential therapeutic target for the treatment of these diseases. Here, we developed one simple ‘in solution’ steady state fluorescence resonance energy transfer (FRET) technique to obtain the dissociation constant (Kd) of the interaction between LFA-1 and ICAM-1. Moreover, we developed the assay into a screening platform to identify peptides and small molecules that inhibit the LFA-1/ICAM-1 interaction. For the FRET pair, we used Alexa Fluor 488-LFA-1 conjugate as donor and Alexa Fluor 555-human recombinant ICAM-1 (D1-D2-Fc) as acceptor. From our quantitative FRET analysis, the Kd between LFA-1 and D1-D2-Fc was determined to be 17.93±1.34 nM. Both the Kd determination and screening assay were performed in a 96-well plate platform, providing the opportunity to develop it into a high-throughput assay. This is the first reported work which applies FRET based technique to determine Kd as well as classifying inhibitors of the LFA-1/ICAM-1 interaction.

Studying DNA looping by single-molecule FRET.

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

Generation of an intramolecular three‐color fluorescence resonance energy transfer probe by site‐specific protein labeling

Fluorescence resonance energy transfer (FRET) is a valuable tool for studying protein structure, folding and interactions. The steep distance dependence of the FRET efficiency requires the donor and acceptor to be in close proximity (1-7.5 nm) to exhibit sufficient energy transfer. One possibility to overcome this limitation is the usage of a FRET cascade that utilizes more than one FRET pair. Essential for realizing this FRET cascade is the site-specific introduction of multiple fluorophores to a given protein, which remains a great challenge. In this study, orthogonal labeling techniques, including fluorescent protein tagging, oxime ligation and kinetically controlled cysteine conjugation, are employed to introduce three fluorophores at specific sites of Rab1b GTPase, yielding a triple-labeled FRET probe. The generated protein probe exhibits efficient energy transfer from the primary donor enhanced green fluorescent protein over the intermediate acceptor rhodamine to the final acceptor Dy630. The labeling strategy opens up a new avenue for multi-color labeling of proteins, facilitating long-distance FRET studies.

Ligand-Dependent Conformational Changes in the Cardiac Ryanodine Receptor

Global conformational changes in the three-dimensional (3D) structure of the calcium release channel/ryanodine receptor (RyR) occur upon ligand activation. Of several ligands that activate RyR, Ca2+ is the primary activator. Although RyR Ca2+ activation is well characterized functionally, little is known about the conformational changes in RyR induced by Ca2+. Here we generated three fluorescence resonance energy transfer (FRET)-based conformational probes. Each of these probes was constructed by inserting a CFP into one domain and a YFP into a neighboring domain in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (CFP/YFP FRET pair). These CFP/YFP FRET pairs were located in the “clamp” region (RyR2S2367-CFP/Y2801-YFP), the calmodulin binding region (RyR2R3595-CFP/K4269-YFP), and the “bridge” region (RyR2S437-YFP/S2367-CFP), respectively. We monitored the conformational changes in these regions by recording the FRET signals, and the extent of Ca2+ release by measuring store Ca2+ depletion in HEK293 cells expressing each of the CFP/YFP FRET pairs upon activation by Ca2+, caffeine, and ATP. Surprisingly, we found that different ligands induced different conformational changes in different regions of RyR2. For instance, we detected conformational changes in the clamp region for caffeine and ATP, but not for Ca2+, although they all induced Ca2+ release. Considering Ca2+ as the primary activator of RyR2, we determined the impact of cytosolic Ca2+ sensing mutation E3987A on conformational changes. Interestingly, this single mutation abolishes caffeine-induced conformational changes, but not caffeine-induced Ca2+ release. These observations demonstrate that conformational changes in RyR2 are ligand-dependent, and that E3987, which is critical for cytosolic Ca2+ sensing, is essential for ligand-induced conformational changes (Supported by CFI, CIHR, HSFC, NIH, and LCIA).

Nabi, a Novel FRET-Based Voltage Sensor Protein

FRET (fluorescence resonance energy transfer) has been used to develop genetically encoded biosensors. FRET-based voltage sensors can be useful to monitor change of membrane potential and neuronal activity because the ratio signal removes noise from a common source such as heart beat artifacts. Positions of fluorescent donor and acceptor in voltage sensor proteins may affect plasma membrane expression, signal intensity, and response time constant of FRET. We developed “Nabi” voltage sensors by using the voltage-sensing domain of CiVSP (Ciona intestinalis voltage sensitive phosphatase) as a backbone. We aimed at improving FRET performance of voltage sensors by optimizing interactions between fluorescent donor and acceptor. For this effort, we designed a series of constructs with insertion of two fluorescent proteins (FPs) at different locations in the CiVSP backbone. We engineered a total of 40 different combinatorial constructs to evaluate 6 different locations for one FP and 10 different locations for the other FP of FRET pairs. By examining FRET signals of these voltage sensors in response to changes in membrane potential in HEK293 cells, we identified amino acid residues of CiVSP at which insertion of FP pairs can generate efficient FRET signals. Nabi1 series of probes contained UKG and mKO (Tsutsui H et al., 2008) as FRET donor and acceptor. Nabi2 series contained Clover and mRuby2 (Lam AJ et al., 2012) as FRET pairs. Nabi voltage sensors had improved signal size (up to 15% ΔF/F) and time constant compared to the previously reported FRET based voltage probes. Nabi2 was able to respond to single action potentials in primary cultured neurons. Supported by US NIH Grants DC005259 and grant WCI 2009-003 from the National Research Foundation of Korea.

Phosphatidylserine and Factor VA Regulate Factor Xa Structure

Human factor Xa (FXa), and its cofactor (FVa), play a key role in blood coagulation by activating prothrombin to thrombin. Phosphatidylserine (PS), exposed on activated platelet membranes, allosterically promotes prothrombin activation by FXa. To gain insight into the structural basis of this allosteric regulation, we used fluorescence resonance energy transfer (FRET) to monitor changes in FXa length in response 1] to soluble PS (dicaproyl-phospahtidylserine; C6PS), 2] to PS membranes, and 3] to FVa in the presence of C6PS and membranes. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa fluor 555) near but not in the membrane. Results demonstrate that FXa structure changes in response to binding of C6PS to two sites, a regulatory site (R) at the N-terminus (previously identified as involving the Gla and EGFn domains) and a presumptive protein-recognition site in the catalytic domain (P). Binding of C6PS to the regulatory site increased the inter-probe distance by ∼ 3 A, while saturation of both sites further increased the distance by ∼ 10 A. FXa binding to a membrane produced a smaller length increase (∼1.6 A), indicating that FXa has somewhat different structures on a membrane and when bound to C6PS in solution. However, when both FVa2 and either C6PS or PS-containing membranes bound to FXa, the overall changes in length were comparable (∼ 5.8 A), indicating that C6PS and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that reported functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with structural regulation demonstrated here. Results further demonstrate the potential usefulness of FRET in analyzing structure-function relationships in FXa and in the FXa.FVa2 complex. Supported by NIH grants HL72827 to BRL and HL43106 to WHK.

Tmfret to Study Short-Range Interactions at the Membrane

Fluorescence resonance energy transfer (FRET) is a technique used to determine the distance between two fluorophores. However, the precision of the distance measurement is limited by the size of the fluorophores and their linkers. In addition, distances over which traditional FRET pairs are sensitive is on the order of the size of many proteins. Thus, short-range interactions have been difficult to study with FRET.Transition Metal Ion FRET (tmFRET) is a novel technique in which the acceptor fluorophore is replaced with a transition metal divalent cation which acts as a non-fluorescent acceptor. The proximity of a fluorescently labeled protein to the metal ions can be determined by the quenching of the donor fluorophore. The small size of the metal ions coupled with the use of small donor fluorphores, such as bimane, allows precise measurement of short-range interactions on the order of 8-20 A.Here we studied the use of tmFRET for measuring interactions at a cell membrane. As proof-of-principle, we used the membrane-resident dye Octadecyl Rhodamine B Chloride (R18) as a FRET donor and metal-bound stearoyl-NTA as a non-fluorescent quencher. Because stearoyl-NTA and R18 both reside in the membrane and have the same acyl chains, we expect the metal-bound NTA group to be well within the distance range of R18 required for tmFRET. Cells were labeled with R18 via transient introduction into the bath. Perfusion of metal-bound stearoyl-NTA into the bath produced quenching of the R18 fluorescence. We studied the reversibility of this effect by subsequently perfusing the cells with EDTA, which chelates the metal ions. Finally, we determined the concentration dependence of R18 quenching by metal-bound stearoyl-NTA. These experiments lay the groundwork for future studies in which tmFRET will be used to measure the distance of fluorophore-tagged proteins from the membrane.

Local Dynamics of FRET Dyes in an Intrinsically Disordered Protein Studied by MD Simulations

Fluorescence resonance energy transfer (FRET) is widely used to track structural changes of biomolecules such as conformational transitions, aggregation or folding. The energy transfer efficiency between a donor-acceptor dye pair is a measure of their mutual separation.However, several assumptions about the dynamics of the dyes have to be made to quantitatively interpret the data measured in single molecule or bulk experiments. In particular, the mutual orientation of their transition dipole vectors determines the FRET efficiency, and has to be disentangled from the inter-dye distance. To this aim, it is usually assumed that both dyes adopt an isotropic orientation and that their orientational fluctuations are fast compared to their excitation decay rate. In this case, an orientation factor of κ2 = 2/3 is obtained.In extended molecular dynamics (MD) simulations we investigated the local flexibility of FRET pairs (tryptophan as donor, coumarin as acceptor) located in the N-terminal and NAC region of alpha-synuclein (aS) which is an entirely intrinsically disordered protein (IDP) of 14.4kD. MD simulations of FRET probes yield instantaneous information about their mutual orientation κ2 and distance RDA which are typically inaccessible to experiments.The orientation factors obtained from our MD simulations deviated considerably from the isotropic value κ2 = 2/3. Further, rotational autocorrelation functions obtained for both dyes reveal a slow diffusion on the surface of the protein. Unexpectedly, we found that slow internal structural fluctuations of aS can cause transient immobilization of one or both dyes, which markedly affects the interpretation of FRET data and might be a general feature of FRET probes attached to IDPs.M. Hoefling, N.Lima, D.Haenni, C.A. M.Seidel, B.Schuler, H.Grubmuller, PLoSONE 6(5): e19791. doi:10.1371/journal.pone.0019791.A.Grupi, E.Haas, JMB, 405:1267-1283 (2011).B.W. van der Meer, Rev.Mol.Biotechnology, 82:181-196 (2002).

Dual-Color Nanoscale Assemblies of Structurally Stable, Few-Atom Silver Clusters, As Reported by Fluorescence Resonance Energy Transfer

We develop approaches to hold fluorescent silver clusters composed of only 10-20 atoms in nanoscale proximity, while retaining the individual structure of each cluster. This is accomplished using DNA clamp assemblies that incorporate a 10 atom silver cluster and a 15 or 16 atom silver cluster. Thermally modulated fluorescence resonance energy transfer (FRET) verifies assembly formation. Comparison to Förster theory, using measured spectral overlaps, indicates that the DNA clamps hold clusters within roughly 5 to 6 nm separations, in the range of the finest resolutions achievable on DNA scaffolds. The absence of spectral shifts in dual-cluster FRET pairs, relative to the individual clusters, shows that select few-atom silver clusters of different sizes are sufficiently stable to retain structural integrity within a single nanoscale DNA construct. The spectral stability of the cluster persists in a FRET pair with an organic dye molecule, in contrast to the blue-shifted emission of the dye.