Dependent Fluorescence Resonance Energy Transfer Research Articles

Spectroscopic and theoretical investigation of conformational changes of proteins by synthesized pyrimidine derivative and its sensitivity towards FRET application

Interest in synthesizing and characterizing (IR, NMR and HRMS spectroscopic methods) a pyrimidine based Schiff-base ligand, 2-(2-(Anthracen-9-ylmethylene) hydrazinyl)-4,6-dimethyl pyrimidine (ANHP) has been developed for its application to ascertain the conformational change of protein and sensitivity towards fluorescence resonance energy transfer (FRET) process. Location of ANHP in bovine serum albumin (BSA) and human serum albumin (HSA) proteins environment has been determined using different spectroscopic techniques. Weakly fluorescent ANHP have shown greater protein induced fluorescence enhancement (PIFE) in case of HSA than BSA, though in both cases energy transfer efficiency are almost same but difference in binding constant values encourages us to find the location of ANHP within the complex protein environment. From the FRET parameter and α-helicity change, it has been found that ANHP bound with Trp-214 of HSA and surface Trp-134 of BSA. Conformational changes of proteins have been observed more for HSA than BSA in presence of ANHP, which has confirmed the location of ANHP in both the protein environments. Coupled with experimental studies, molecular docking analysis has also been done to explain the locations and distance dependent FRET process of ANHP in both proteins.

Tuning of donor-acceptor linker in rhodamine-coumarin conjugates leads remarkable solvent dependent FRET efficiency for Al3+ imaging in HeLa cells

A series of rhodamine-coumarin conjugates with tunable linkers function as FRET based sensors for biological Al3+ imaging in living HeLa cells under fluorescence microscope. In methanol, all probes show chelation enhanced fluorescence (CHEF) assisted fluorescence resonance energy transfer (FRET) process. A remarkable change in FRET efficiency is observed with increasing water content of the media for a particular probe. DFT studies corroborate experimental observations.

The effect of mouse twinfilin-1 on the structure and dynamics of monomeric actin

The effect of twinfilin-1 on the structure and dynamics of monomeric actin was investigated with fluorescence spectroscopy and differential scanning calorimetry experiments. Fluorescence anisotropy measurements proved that G-actin and twinfilin-1 could form a complex. Due to the formation of the complexes the dissociation of the nucleotide slowed down from the nucleotide-binding pocket of actin. Fluorescence quenching experiments showed that the accessibility of the actin bound ε-ATP decreased in the presence of twinfilin-1. Temperature dependent fluorescence resonance energy transfer and differential scanning calorimetry experiments revealed that the protein matrix of actin becomes more rigid and more heat resistant in the presence of twinfilin-1. The results suggest that the nucleotide binding cleft shifted into a more closed and stable conformational state of actin in the presence of twinfilin-1.

Dynamics of the Cytoplasmic Region of an AMPA-Subtype Glutamate Receptor Revealed by State Dependent FRET

AMPA receptors (AMPARs) are glutamate-gated ion channels, which mediate fast excitatory neurotransmission in the central nervous system. Extensive crystallographic studies of the extracellular domains and, more recently, crystal structures and single particle EM reconstructions of full-length receptors have set the framework for further investigations of receptor conformational dynamics, gating mechanism and regulation. However, intracellular regions are either truncated or not resolved in these structures. Further, the conformational transitions of the intracellular domains during receptor gating have not been investigated.In present study, we have explored single and double fusions of cyan and yellow variants of green fluorescent protein (CFP and YFP, respectively) at intracellular sites of AMPAR to enable measurement of conformational changes using Fluorescence Resonance Energy Transfer (FRET) in live cells. The fluorescent fusions retain wild-type receptor expression and kinetic properties. Fluorescence Lifetime Imaging (FLIM) showed ligand-dependent FRET efficiency. Conformational rearrangements accompanying receptor function were measured using a Patch Clamp Fluorometry (PCF) setup on live HEK 293 cells in real time. Our results suggest that FRET efficiency is dependent on the functional state of the receptor and allosteric modulation by Cyclothiazide, an AMPA receptor desensitisation blocker. Thus the intracellular sites undergo conformational rearrangements during receptor function.

Sensing of Hg+2 and Ag+ through a pH dependent FRET system: Fabrication of molecular logic gates

The fluorescence resonance energy transfer (FRET) from the donor 6,7-dimethoxy-2,4[1H,3H]-quinazolinedione (Q) to the acceptor lumichrome (L) exhibits strong pH dependency and in aqueous solution at pH 9 it is used to sense Hg+2 or Ag+ ions, selectively. QL hybrid forms organic nanoparticles through self-assembly at pH 9 as evident from TEM micrographs. The QL system can sense Hg+2 or Ag+ ion in aqueous medium at pH9 at a nanomolar concentration range using FRET pathway via complexation and nanoparticle formation, respectively. In the QL system, the ions can be easily distinguished with the help of UV–vis spectroscopic study or through the naked eye. We have successfully utilized the above metal ions for the fabrication of NOR and INHIBIT molecular logic gates using FRET of QL system as a signal transducer and Hg+2, Ag+ ion and pH as chemical activators.

Fluorescence Resonance Energy Transfer in Microemulsions Composed of Tripled-Chain Surface Active Ionic Liquids, RTILs, and Biological Solvent: An Excitation Wavelength Dependence Study

In this article we have reported the fluorescence resonance energy transfer (FRET) study in our earlier characterized surface active ionic liquids (SAILs)-containing microemulsion, i.e., N-methyl-N-propylpyrrolidinium bis(trifluoromethanesulfonyl)imide ([P13][Tf2N])/[CTA][AOT]/isopropyl myristate ([IPM]) and N,N,N-trimethyl-N-propylammonium bis(trifluoromethanesulfonyl)imide ([N3111][Tf2N])/[CTA][AOT]/[IPM] microemulsions (Banerjee, C.; Mandal, S.; Ghosh, S.; Kuchlyan, J.; Kundu, N.; Sarkar, N. J. Phys. Chem. B 2013, 117, 3927-3934). The occurrence of effective FRET from the donor, coumarin-153 (C-153) to the acceptor rhodamine 6G (R6G) is evident from the decrease in the steady state fluorescence intensity of the donor with addition of acceptor and subsequent increase in the fluorescence intensity of the acceptor in the presence of donor. The excitation wavelength dependent FRET from C-153 to R6G has also been performed to assess the dynamic heterogeneity of these confined systems. In time-resolved experiments, the significant rise time of the acceptor in the presence of the donor further confirms the occurrence of FRET. The multiple donor-acceptor (D-A) distances, for various microemulsions, obtained from the rise times of the acceptor emission in the presence of a donor can be rationalized from the varying distribution of the donor, C-153, in the different regions of the microemulsion. Time-resolved measurement reveals that with increasing excitation wavelength from 408 to 440 nm, the contribution of the faster rise component of FRET increases significantly due to the close proximity of the C-153 and R6G in the polar region of the microemulsion where occurrence of FRET is very high. Moreover, we have also studied the FRET with variation of R (R = [room temperature ionic liquids (RTILs)]/[surfactant]) and shown that the effect of excitation wavelength on FRET is similar irrespective of R values.

The effect of ADF/cofilin and profilin on the dynamics of monomeric actin

The main goal of the work was to uncover the dynamical changes in actin induced by the binding of cofilin and profilin. The change in the structure and flexibility of the small domain and its function in the thermodynamic stability of the actin monomer were examined with fluorescence spectroscopy and differential scanning calorimetry (DSC). The structure around the C-terminus of actin is slightly affected by the presence of cofilin and profilin. Temperature dependent fluorescence resonance energy transfer measurements indicated that both actin binding proteins decreased the flexibility of the protein matrix between the subdomains 1 and 2. Time resolved anisotropy decay measurements supported the idea that cofilin and profilin changed similarly the dynamics around the fluorescently labeled Cys-374 and Lys-61 residues in subdomains 1 and 2, respectively. DSC experiments indicated that the thermodynamic stability of actin increased by cofilin and decreased in the presence of profilin. Based on the information obtained it is possible to conclude that while the small domain of actin acts uniformly in the presence of cofilin and profilin the overall stability of actin changes differently in the presence of the studied actin binding proteins. The results support the idea that the small domain of actin behaves as a rigid unit during the opening and closing of the nucleotide binding pocket in the presence of profilin and cofilin as well. The structural arrangement of the nucleotide binding cleft mainly influences the global stability of actin while the dynamics of the different segments can change autonomously.

A Novel Ionic Liquid-in-Oil Microemulsion Composed of Biologically Acceptable Components: An Excitation Wavelength Dependent Fluorescence Resonance Energy Transfer Study

In this work we have reported the formulation of a novel ionic liquid-in-oil (IL/O) microemulsion where the polar core of the ionic liquid, 1-ethyl-3-methylimidazolium n-butylsulfate ([C2mim][C4SO4]), is stabilized by a mixture of two nontoxic nonionic surfactants, polyoxyethylene sorbitan monooleate (Tween-80) and sorbitan laurate (Span-20), in a biological oil phase of isopropyl myristate (IPM). The formation of the microemulsion droplets has been confirmed from the dynamic light scattering (DLS) and phase behavior study. To assess the dynamic heterogeneity of this tween-based IL/O microemulsion, we have performed an excitation wavelength dependent fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to rhodamine 6G (R6G). The multiple donor-acceptor (D-A) distances, ∼15, 30, and 45 Å, obtained from the rise times of the acceptor emission in the presence of a donor can be rationalized from the varying distribution of the donor, C480, in the different regions of the microemulsion system. With increasing the excitation wavelength from 375 to 408 nm, the contribution of the rise component of ∼240 ps which results the D-A distance of ∼30 Å increases significantly due to the enhanced contribution of the C480 probe molecules closer to the acceptor in the ionic liquid pool of the microemulsion.

Donor-to-Acceptor Distance Dependent Fluorescence Resonance Energy Transfer Efficiency for Multiple Donors and Acceptors System Confined within 2-Dimensional Fluid of Supported Lipid Bilayer

We study experimentally the dependence of fluorescence resonance energy transfer (FRET) efficiency on the distance between donors and acceptors that are embedded in a supported lipid bilayer. The FRET efficiencies are determined by comparing the donor fluorescence intensities before and after acceptor photobleaching. The obtained data are compared with a theory that considers multiple donors and multiple acceptors existing in a two-dimensional field. We achieve good qualitative agreement in terms of the relationship between the FRET efficiency and the donor-to-acceptor distances. The Forster radius is also estimated from the comparison, which shows good quantitative agreement. The significance of the present demonstration is in showing that acceptor photobleaching experiments can quantitatively evaluate the FRET phenomenon, which indicates that the supported lipid bilayer behaves as an ideal two-dimensional fluidic system for embedded molecules. [DOI: 10.1380/ejssnt.2012.121]

Analytical Nanosphere Sensors Using Quantum Dot−Enzyme Conjugates for Urea and Creatinine

An enzyme-linked analytical nanosphere sensor (ANSor) is described, responding to enzyme-substrate turnover in the vicinity of a quantum dot (QD) due to coimmobilized enzyme and pH sensitive ligand. QD capping by mercapto-alkanoic acids were rejected as a pH sensitive ligand, but with the use of a layer-by-layer assembly on mercaptopropionic capped QDs and an intermediate poly(allylamine hydrochloride) layer, anthraquinone sulfonate (calcium red, CaR) was introduced to modify the pKa in the immobilized system > 8. QD-CaR absorption shows spectral overlap with QD530 emission at all pHs and gives a complex pH dependent fluorescence resonance energy transfer (FRET) efficiency, due to excited state proton transfer (λ(ex) = 540 nm; λ(em) = 585 nm). In contrast QD615-CaR with spectral overlap between the QD and CaR gave a strong and reproducible pH response. QD-urease and QD-creatinine deiminase conjugates could be linked with pH changes produced by enzyme degradation of urea and creatinine, respectively. Close coupling between the pH sensitive QD and enzyme conjugate maximized signal compared with solution based assays: QD-urease and QD-CD bioconjugates were tested in model biological media (Dulbecco's modified Eagle's Medium and fetal calf serum) and in urine, showing a response in 3-4 min.

DNA-Directed Assembly of Supramolecular Fluorescent Protein Energy Transfer Systems

Fluorescent proteins with a wide variety of physicochemical properties have evolved in the past few years. The use of these proteins for applications in biomolecular nanosciences requires their precise positioning at the nanometer length scale. To address this challenge, we report here on the self-organization of DNA-tagged fluorescent probes to construct a set of photofunctional supramolecular complexes which include the enhanced yellow fluorescent protein (EYFP). The optical functionality is based on the strongly distance dependent fluorescence resonance energy transfer (FRET), occurring between the donor (EYFP) and an acceptor fluorophore, i.e., the fluorescent dye Atto647. The photophysical properties of four bimolecular FRET complexes, each possessing a well-defined donor-acceptor distance defined by the length of the interconnecting DNA backbone, are investigated by two-dimensional photoluminescence excitation spectroscopy (2D-PLE).

The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments

In this work the effect of phalloidin and jasplakinolide on the dynamic properties and thermal stability of actin filaments was studied. Temperature dependent fluorescence resonance energy transfer measurements showed that filaments of Ca-actin became more rigid in the presence of phalloidin or jasplakinolide. Differential scanning calorimetric data implied that the stiffer filaments also had greater thermal stability in the presence of phalloidin or jasplakinolide. The fluorescence and calorimetric measurements provided evidences that the extent of stabilization by jasplakinolide was greater than that by phalloidin.

Single-molecule studies highlight conformational heterogeneity in the early folding steps of a large ribozyme.

The equilibrium folding of the catalytic domain of Bacillus subtilis RNase P RNA is investigated by single-molecule fluorescence resonance energy transfer (FRET). Previous ensemble studies of this 255-nucleotide ribozyme described the equilibrium folding with two transitions, U-to-I(eq)-to-N, and focused on the I(eq)-to-N transition. The present study focuses on the U-to-I(eq) transition. Comparative ensemble measurements of the ribozyme construct labeled with fluorescein at the 5' end and Cy3 at the 3' end show that modifications required for labeling do not interfere with folding and help to define the Mg(2+) concentration range for the U-to-I(eq) transition. Histogram analysis of the Mg(2+)-dependent single-molecule FRET efficiency reveals two previously undetermined folding intermediates. The single-molecule FRET trajectories exhibit non-two-state and nonergodic behaviors at intermediate Mg(2+) concentrations on the time scale of seconds. The trajectories at intermediate Mg(2+) concentrations are classified into five classes based on three FRET levels and their dynamics of interconversion within the measured time range. This heterogeneity, together with the observation of "nonsudden jump" FRET transitions, indicates that the early folding steps of this ribozyme involve a series of intermediates with different degrees of kinetic isolation and that folding occurs under kinetic control and involves many "local" conformational switches. A free energy contour is constructed to illustrate the complex folding surface.

Surface Pressure Dependent Fluorescence Resonance Energy Transfer in Mixed Monolayers of Amphiphilic Coumarin and Texas Red at the Air−Water Interface

This paper reports the spectroscopic characteristics of pure N-(Texas Red sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (TR) and 3-(2-benzothiaxolyl)-4-cyano-7-(octadecyloxy)coumarin (BCOC) at the air−water interface. Absorption and steady state fluorescence studies of these dyes at the air−water interface reveals the formation of aggregates at the air−water interface. Detailed studies revealed the formation of J-aggregates in the case of TR and both J- and H-aggregates in the case of BCOC. Polarized fluorescence studies confirmed that the dye molecules were highly oriented at the air−water interface. Surface pressure dependent fluorescence emission studies showed extensive quenching of the TR fluorescence that has been attributed to an efficient energy transfer from the monomeric species of TR to their aggregates which are nonfluorescent. In the case of BCOC, fluorescence emission occurs only from the J-aggregates of BCOC as the H-aggregates are nonfluorescent. Mixed films of BCOC and TR with DPPC show unique behavior. Even at very low concentration of TR, the BCOC fluorescence is completely quenched and the sensitized fluorescence from TR dominate, thus indicating efficient energy transfer from BCOC to the TR moieties. With an increase in the surface pressure, the same features are found to predominate and the quenching of TR fluorescence is attributed to the generation of TR aggregates caused as a result of compressing the monolayer. At higher surface pressures, the characteristics of the TR fluorescence are found to disappear and the fluorescence characteristics of BCOC dominate. One explanation of such behavior could be the deactivation of BCOC via the radiative pathway and inefficent energy transfer from the aggregates of BCOC to the aggregates of TR. This study therefore establishes that FRET can be a useful tool for studying aggregation in supramolecular LB assemblies.