Forster Resonance Energy Transfer Studies Research Articles

Single-Molecule Characterization of Cy3.5 -Cy5.5 Dye Pair for FRET Studies of Nucleic Acids and Nucleosomes

Single molecule FRET (Forster resonance energy transfer) is very powerful method for studying biomolecular binding dynamics and conformational transitions. Only a few donor - acceptor dye pairs have been characterized for use in single-molecule FRET (smFRET) studies. Hence, introducing and characterizing additional FRET dye pairs is important in order to widen the scope of applications of single-molecule FRET in biomolecular studies. Here we characterize the properties of the Cy3.5 and Cy5.5 dye pair under FRET at the single-molecule level using naked double-stranded DNA (dsDNA) and the nucleosome. We show that this pair of dyes is photostable for ~ 5 min under continuous illumination. We also report Cy3.5-Cy5.5 FRET proximity dependence and stability in the presence of several biochemical buffers and photoprotective reagents in the context of double-stranded DNA. Finally, we demonstrate compatibility of the Cy3.5-Cy5.5 pair for smFRET in vitro studies of nucleosomes.

FRET Study Between Carbon Quantum Dots and Malachite Green by Steady-State and Time-Resolved Fluorescence Spectroscopy

Background: Understanding the interaction between different organic dyes and carbon quantum dots helps us to understand several photo physical processes like electron transfer, energy transfer, molecular sensing, drug delivery and dye degradation processes etc. Objective: The primary objective of this study is to whether the carbon quantum dots can act as an electron donor and can participate in the different photo physical processes. Methods: In this work, Carbon Quantum Dots (CQDLs) are synthesized in most economical and simple carbonization method where petals of Nelumbo nucifera L. are used as a carbon precursor. The synthesized CQDLs were characterized by using experimental techniques like UV−Vis absorption, FT-IR, Transmission Electron Microscopy (TEM), steadystate and time-resolved fluorescence spectroscopy. Results: The spectral analysis shows that the so synthesized CQDLs are spherical in shape and its diameter is around 4.2 nm. It shows the fluorescence emission maximum at 495 nm with a quantum yield of 4%. In this work the interaction between Carbon Quantum Dots (CQDLs) and an organic dye Malachite Green (MG) is studied using fluorescence spectroscopic technique under ambient pH condition (At pH 7). The quenching mechanism of CQDLs with MG was investigated using Stern-Volmer equation and time-resolved fluorescence lifetime studies. The results show that the dominant process of fluorescence quenching is attributed to Forster Resonance Energy Transfer (FRET) having a donor acceptor distance of 53 Å where CQDLs act as a donor and MG acts as an acceptor. Conclusion: This work has a consequence that CQDLs can be used as a donor species for different photo physical processes such as photovoltaic cell, dye sensitized solar cell, and also for antioxidant activity study.

Accurate Determination of the RNA Junctions via Single-Molecule High-Precision FRET Measurements

Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is a powerful tool for analyzing the biomolecular structure. We apply multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting measurements (eTCSPC) to perform FRET study on RNA three- and four-way-junctions (4WJs and 3WJs) which are derived from the hairpin ribozyme.Overall 283 FRET pairs were measured with single-molecule MFD and analyzed with the analysis toolkit [1] that includes probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation.In order to study the influence of the junction on the RNA structure we studied the functional junction part with prolonged helices. Bulge and sequence variations were considered as dominant factors influencing junction conformations for RNA 3WJ. Six different RNA 3WJ with different sequences were studied, two of which have two and five unpaired nucleotides in the junction region.We found three different conformers for RNA 4WJ. However RNAs 3WJ have only one predominant conformer. Furthermore we report that bulges in the junction region determine orientation and rotation of helices, inducing coaxial stacking. Noteworthy the stacked helices are different for the 3WJs with different bulges. Our results show that small changes in the sequence make dramatic changes in RNA 3WJ tertiary structures which are expected to have significant impact on the functionality.[1] Kalinin, S. et al, Nature Methods, 9, 1218-1225 (2012).

Color-Tunable Fluorescence and White Light Emission from Mesoporous Organosilicas Based on Energy Transfer from 1,8-Naphthalimide Hosts to Perylenediimide Guests

The present work reports Forster resonance energy transfer (FRET) from 1,8-naphthalimide (NI) donors bound to the pore walls of mesoporous silicas to perylenediimide (PDI) acceptors doped into the mesochannels. Mesoporous organosilicas containing covalently bound NI were synthesized by co-condensation of tetraethylorthosilicate (TEOS) with N-(3-(triethoxysilyl)propyl)-1,8-naphthalimide (TEPNI) in the presence of a block copolymer surfactant as a template. The resulting materials were highly ordered, presenting a 2D hexagonal structure, and displayed easily tunable optical properties, which could be controlled by the amount of NI in the sample. A sample prepared from a diluted TEPNI solution (SBANId) presented a blue, monomerlike emission. In contrast, when a concentrated TEPNI solution was used, the resulting material (SBANIc) displayed a green, excimerlike emission. For the FRET studies, N,N′-bis(2,6-dimethylphenyl)-3,4,9,10-perylenediimide was doped into the pores of the SBANI samples from chloroform so...

RNA Junctions Structure and Distance Determination via Accurate Single-Molecule High-Precision FRET Measurements

Forster-Resonance-Energy-Transfer (FRET) restrained high-precision structural modeling is a powerful tool for analyzing biomolecular structures. Here we apply multi-parameter fluorescence detection (MFD) of single molecules and ensemble Time-Correlated Single Photon Counting measurements to perform FRET study on RNA three- and four-way-junctions (4WJs and 3WJs) with a high level of precision in distance better than 1% of the Forster radius [1].We have generated a database of RNA 4WJs and six different RNA 3WJs with different bulges and sequences to study the influence of these factors on the junction conformations of RNA 3WJs. Overall 260 FRET pairs were measured with single-molecule MFD at 20 mM MgCl2 concentration and analyzed with the analysis toolkit [2] that includes probability distribution analysis (PDA) for FRET distance determination and FRET position and screening (FPS) toolkit for structural model generation.Monte Carlo simulations showed that sterically allowed conformational space for RNA junctions is large. However, FRET measurements detect the existence of three different static conformers for RNA 4WJ, whereas RNA 3WJs have only one predominant static conformation. terically allowed conformational space for RNA is large. Their junction geometry was described in terms of mutual and Euler angles between helices. The FRET-derived structures suggest that the sequence dictates a junction specific conformation within the large topology space. Furthermore we see that bulges in the junction region determine orientation and rotation of helices and induce coaxial stacking between two of them.[1] Antonik, M., et al., J.Phys.Chem.B, 110, 6970-6978 (2006)[2] Kalinin, S. et al, Nat. Meth., 9, 1218-1225 (2012)

How Histone Modifications Change Nucleosome Stability – FRET Studies on Single Molecules and in Bulk

Using Forster resonance energy transfer (FRET) measurements in bulk and on single molecules, we assessed the structural role of histone acetylation and of some point mutations in nucleosomes reconstituted on the 170bp long Widom 601 DNA sequence. By measuring distances between fluorescently labeled parts of the nucleosome we could follow the salt-induced nucleosome disassembly, using donor-acceptor pairs on the ends or in the internal part of the nucleosomal DNA, and on histones. The single molecule observations helped to characterize the intermediate states as well as the distributions of the populations [1,2].

Three-Dimensional Solution-Phase Förster Resonance Energy Transfer Analysis of Nanomolar Quantum Dot Bioconjugates with Subnanometer Resolution

Luminescent semiconductor quantum dots (QDs) play an important role in optical biosensing and, in particular, in FRET (Forster resonance energy transfer)-based luminescent probes. The QD materials that form the basis for these probes are in actuality quite heterogeneous and consist of different types of QDs with variations in material compositions, surface coatings, and available biofunctionalization strategies. To optimize their role in active sensors that rely on FRET, extensive physicochemical characterization is required. A technique that can provide precise information about size, shape, and bioconjugation properties of different QD–biomolecule conjugates from a single sample and measurement under actual experimental biosensing conditions would therefore be highly important for advancing QDs to a next generation nanobiosensing tool. Here, we present a detailed FRET study on a large set of QD–biomolecule conjugates, which allows for a homogeneous solution-phase size, shape, and bioconjugation analysis...

Cross Regulation in Signaling Networks: A FRET Study of the PhoB-PhoR Two-Component System in E-Coli

The signal for the PhoR-PhoB two-component regulatory system of E. coli is environmental inorganic phosphate (Pi). A signal for environmental Pi insufficiency is propagated across the membrane to the cytosolic domain of the signalling histidine kinase PhoR via the ABC (ATP-binding cassette) transporter, the Pst (Pi-specific transport) system, in the absence of transport by protein-protein interactions, both in the membrane and the cytoplasm of the cell. Cross regulation also exists between PhoR-PhoB two-component system and key regulatory systems in E. coli, including the ArcB-ArcA two-component system for redox signalling. Such cross regulation among key signalling systems is thought to be important for integration of diverse cellular pathways in the control of cell growth and for survival under conditions of stress. Knowledge of how these interactions is brought about necessary to understand fully the dynamics of the response of the Pi signalling network to environmental Pi insufficiency and to understand interactions of the PhoR-PhoB and other signalling systems. Here we have employed Forster resonance energy transfer (FRET) techniques (acceptor photobleaching and sensitized emission) coupled with specific knock-out mutants to investigate in live E. coli cells interactions between ECFP-labelled members of the PhoR-PhoB system and protein targets with which they are thought to interact such as the o-succinylbenzoyl-CoA synthase, MenC. We will show a strong enhancement of the FRET signal through the use of protein fusions and knock-out mutants. We will discuss results of this study in terms of their implication on the dynamics of the PhoR-PhoB two-component signal transduction system.

Interaction of the Anticancer Plant Alkaloid Sanguinarine with Bovine Serum Albumin

BackgroundInteraction of the iminium and alkanolamine forms of sanguinarine with bovine serum albumin (BSA) was characterized by spectroscopic and calorimetric techniques.Methodology/Principal FindingsFormation of strong complexes of BSA with both iminium and alkanolamine forms was revealed from fluorescence quenching of sanguinarine. Binding parameters calculated from Stern-Volmer quenching method revealed that the neutral alkanolamine had higher affinity to BSA compared to the charged iminium form. Specific binding distances of 3.37 and 2.38 nm between Trp 212 (donor) and iminium and alkanolamine forms (acceptor), respectively, were obtained from Forster resonance energy transfer studies. Competitive binding using the site markers warfarin and ibuprofen, having definite binding sites, demonstrated that both forms of sanguinarine bind to site I (subdomain IIA) on BSA. Sanguinarine binding alters protein conformation by reducing the α-helical organization and increasing the coiled structure, indicating a small but definitive partial unfolding of the protein. Thermodynamic parameters evaluated from isothermal titration calorimetry suggested that the binding was enthalpy driven for the iminium form but favoured by negative enthalpy and strong favourable entropy contributions for the alkanolamine form, revealing the involvement of different molecular forces in the complexation.Conclusions/SignificanceThe results suggest that the neutral alkanolamine form binds to the protein more favourably compared to the charged iminium, in stark contrast to the reported DNA binding preference of sanguinarine.

Single Molecule Fluorescence Studies of Antibody Structure

The structures of antibodies have been examined using a variety of techniques. Forster resonance energy transfer (FRET) is one technique well-suited for the study of these highly flexible proteins. We will present results from single molecule FRET studies of the distribution of distances between the two-antigen binding sites of an IgG antibody. The antibody studied is the catalytic aldolase antibody 38C2 (Wagner, J.; Lerner, R. A.; Barbas, C. F. (1995) Science270, 1797). This antibody was chosen because it is known to covalently bind a diketone hapten. By linking donor and acceptor dye molecules to the diketone hapten, the antigen-binding sites were selectively labeled for FRET studies. The FRET measurements involved the examination of photon bursts from freely diffusing donor-acceptor labeled antibodies, from which a histogram of the conformations present was constructed.

Förster's resonance energy transfer between Fullerene C 60 and Coumarin C440

The interaction between Coumarin C440 with Fullerene C 60 has been studied by fluorescence and time resolved spectroscopic techniques. The Coumarin C440–Fullerene C 60 pair shows Forster's resonance energy transfer (FRET) from Coumarin C440 (donor) to Fullerenes C 60 (acceptor). The FRET efficiency of this pair increases with the increase of the acceptor concentration. The critical energy transfer distance ( R 0) at which transfer efficiency is 50% is found to be 34 Ǻ. Stern–Volmer plot indicates static as well as dynamic quenching. However, the FRET studies show highest efficiency at the critical stage of dimer formation.

Binding to and photo-oxidation of cardiolipin by the phthalocyanine photosensitizer Pc 4

Cardiolipin is a unique phospholipid of the mitochondrial inner membrane. Its peroxidation correlates with release of cytochrome c and induction of apoptosis. The phthalocyanine photosensitizer Pc 4 binds preferentially to the mitochondria and endoplasmic reticulum. Earlier Förster resonance energy transfer studies showed colocalization of Pc 4 and cardiolipin, which suggests cardiolipin as a target of photodynamic therapy (PDT) with Pc 4. Using liposomes as membrane models, we find that Pc 4 binds to cardiolipin-containing liposomes similarly to those that do not contain cardiolipin. Pc 4 binding is also studied in MCF-7c3 cells and those whose cardiolipin content was reduced by treatment with palmitate. Decreased levels of cardiolipin are quantified by thin-layer chromatography. The similar level of binding of Pc 4 to cells, irrespective of palmitate treatment, supports the lack of specificity of Pc 4 binding. Thus, factors other than cardiolipin are likely responsible for the preferential localization of Pc 4 in mitochondria. Nonetheless, cardiolipin within liposomes is readily oxidized by Pc 4 and light, yielding apparently mono- and dihydroperoxidized cardiolipin. If similar products result from exposure of cells to Pc 4-PDT, they could be part of the early events leading to apoptosis following Pc 4-PDT.

Spectroscopic Characterization of Depolarization Via FRET in a BFP-GFP BoNT/A Assay

The assay is based on depolarization due to Forster Resonance Energy Transfer (FRET) between Blue Fluorescent Protein (BFP) and Green Fluorescent Protein (GFP) moieties linked by a peptide containing residues 134-206 of SNAP-25, the protein substrate for BoNT/A's proteolytic activity. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is −0.04 due to depolarization following FRET from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the FRET distance, the polarization increases significantly to ∼0.4, due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows for an assay (termed DARET for Depolarization After Resonance Energy Transfer) that is robust and sensitive. In this report we characterize the spectroscopic parameters of the system before and after substrate cleavage, including excitation and emission spectra, polarization and time-resolved methods. Our results suggest that the donor and acceptor dipoles are at a large angle (72°) with respect to one another but that the BFP and GFP are in direct contact in the intact substrate. Evidence is also provided to demonstrate the direct interaction of BFP and GFP after cleavage. We have used this assay to determine the enzyme kinetic parameters (Km, Kcat and Vmax) for BoNT/A proteolysis of the assay substrate. Our conclusions bear on the issue of the common choice of 2/3 for κ2 for FRET studies in general and on fluorescent proteins in particular. This work was supported by Allergan Inc.

Cellular Basis Of Nicotine-induced nAChr Upregulation

The upregulation and preferential assembly of high sensitivity (α4)2(β2)3 neuronal nicotinic acetylcholine receptors (nAChRs) contribute to aspects of nicotine addiction such as sensitization and tolerance. The cellular mechanisms of these events, however, remain elusive. We employed fluorescently tagged nAChRs to study plasma membrane upregulation by total internal reflection fluorescence microscopy (TIRFM) as well as changes in intracellular receptor stoichiometry using pixel-based Forster's resonance energy transfer (FRET). To delineate the effect of β2 on α4 nAChR trafficking in the absence of nicotine, mouse neuroblastoma (N2a) cells were transiently transfected with either α4-meGFP/wildtype β2 or α4-meGFP/wildtype β4 subunits (m = monomeric; e = enhanced) and imaged at 48 h post-transfection by TIRFM. To set TIRFM parameters, cells were co-transfected with the pCS2-mcherry plasmid, which served as a reference probe. pCS2-mcherry expresses mcherry with a lyn kinase membrane localization signal, allowing visualization of the PM using red emission from mcherry. The α4-meGFP reporter was used to detect receptor expression at the PM. Results showed that the α4-meGFP/wildtype β2 receptors trafficked to the PM in ∼10 % of the cells while ∼90 % of imaged cells displayed α4-meGFP/wildtype β4 at the PM. In the presence of nicotine (0.1 μM for 48 h), α4-meGFP/wildtype β2 transfected N2a cells displayed a clear increase in receptor trafficking to the PM when visualized using TIRFM. Pixel-based sensitized emission FRET studies on N2a cells transiently transfected with an α4-mcherry and β2-meGFP FRET pair showed that chronic nicotine exposure (1 μM, 24 h) resulted in an increase in assembly of the high sensitivity (α4)2(β2)3 population of receptors, a phenomenon that was blocked by co-incubation with the competitive nAChR antagonist, DhβE (1 μM). These preliminary results point to a modulatory role of β2 subunits as well as a possibly important role for activity-dependent receptor upregulation.

Single-molecule Forster resonance energy transfer study of protein dynamics under denaturing conditions.

Proteins are highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of denaturants, the heterogeneity is greatly enhanced, and fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways. Here, we have studied the structure and dynamics of the small enzyme ribonuclease HI (RNase H) in the presence of the chemical denaturant guanidinium chloride (GdmCl) using single-molecule fluorescence microscopy, with a particular focus on the characterization of the unfolded-state ensemble. A dye pair was specifically attached to the enzyme to measure structural changes through Förster resonance energy transfer (FRET). Enzyme immobilization on star-polymer surfaces that were specially developed for negligible interaction with folded and unfolded proteins enabled us to monitor conformational changes of individual proteins for several hundred seconds. FRET efficiency histograms were calculated from confocal scan images. They showed an expansion of the unfolded proteins with increasing GdmCl concentration. Cross-correlation analysis of donor and acceptor fluorescence intensity time traces from single molecules revealed reconfiguration of the polypeptide chain on a timescale of approximately equal to 20 micros at 1.7 M GdmCl. Slow conformational dynamics gave rise to characteristic, stepwise FRET efficiency changes. Transitions between folded and unfolded enzyme molecules occurred on the 100-s timescale, in excellent agreement with bulk denaturation experiments. Transitions between unfolded conformations were more frequent, with characteristic times of approximately equal to 2 s. These data were analyzed to obtain information on the free energy landscape of RNase H in the presence of chemical denaturants.