Fluorescence Resonance Energy Transfer Efficiency Research Articles

FLIM-FRET Measurements of Voltage Dependent Conformational Changes in the Outer Hair Cell Motor Protein Prestin

Cochlear sensory cells, outer hairs cells (OHCs), elongate and contract in response to changes in transmembrane potential. This electromotility is important for auditory frequency selectivity and sensitivity and is believed to be driven by a motor protein, prestin, which is highly expressed within the membrane of OHCs. Experiments have shown that prestin undergoes self-association leading to the observation that oligomerization might be important for proper function. We have previously utilized acceptor photobleach fluorescence resonance energy transfer (FRET) to study pretin-prestin interactions in living cells. However, traditional FRET, based upon detection of fluorescence intensity, is inherently subjected to artifacts that arise from variations in excitation intensity, photobleaching, and fluorophore concentration. We have recently implemented fluorescence lifetime imaging microscopy (FLIM) to perform robust FLIM-FRET measurements in HEK cells cotransfected with prestin-TFP and prestin-YFP. FRET efficiency was calculated by fitting the lifetime decay of prestin-TFP in cells clamped at various holding potentials. High FRET efficiencies were measured at hyperpolarized potentials, and low FRET efficiencies were measured at depolarized. The results indicated that changes in the transmembrane potential induce a conformational change in prestin that can be detected by FLIM-FRET. Thus, FLIM-FRET can provide a sensitive assay for the mechanical correlate of prestin function.

Solvent dependent competition between fluorescence resonance energy transfer and through bond energy transfer in rhodamine appended hexaphenylbenzene derivatives for sensing of Hg2+ ions

Hexaphenylbenzene (HPB) derivatives 5 and 7 having rhodamine B moieties have been designed and synthesized, and have been shown to display solvent dependent. Fluorescence resonance energy transfer (FRET) and through bond energy transfer (TBET) in the presence of Hg(2+) ions among the various cations (Cu(2+), Pb(2+), Zn(2+), Ni(2+), Cd(2+), Ag(+), Ba(2+), Mg(2+), K(+), Na(+), and Li(+)) have been tested. Derivative 5 displays quite high through bond energy transfer efficiency in the presence of Hg(2+) ions in methanol whereas derivative 7 exhibits better FRET efficiency in the presence of Hg(2+) ions in THF and CH(3)CN than derivative 5.

Fate of paclitaxel lipid nanocapsules in intestinal mucus in view of their oral delivery

The bioavailability of paclitaxel (Ptx) has previously been improved via its encapsulation in lipid nanocapsules (LNCs). In this work, the interactions between LNCs and intestinal mucus are studied because they are viewed as an important barrier to successful oral delivery. The rheological properties of different batches of pig intestinal mucus were studied under different conditions (the effect of hydration and the presence of LNCs). Fluorescence resonance energy transfer (FRET) was used to study the stability of LNCs in mucus at 37°C for at least 3 hours. Diffusion through 223, 446, and 893 μm mucus layers of 8.4, 16.8, and 42 μg/mL Ptx formulated as Taxol® (Bristol-Myers Squibb, Rueil-Malmaison, France) or encapsulated in LNCs (Ptx-LNCs) were investigated. The effect of the size of the LNCs on their diffusion was also investigated (range, 25–110 nm in diameter). Mucus behaves as a non-Newtonian gel with rheofluidifying properties and a flow threshold. The viscous (G″) and elastic (G′) moduli and flow threshold of the two mucus batches varied with water content, but G′ remained below G″. LNCs had no effect on mucus viscosity and flow threshold. The FRET efficiency remained at 78% after 3 hours. Because the destruction of the LNCs would lead to a FRET efficiency below 25%, these results suggest only a slight modification of LNCs after their contact with mucus. The diffusion of Taxol® and Ptx-LNCs in mucus decreases if the mucus layer is thicker. Interestingly, the apparent permeability across mucus is higher for Ptx-LNCs than for Taxol® for drug concentrations of 16.8 and 42 μg/mL Ptx (P<0.05). The diffusion of Ptx-LNCs through mucus is not size-dependent. This study shows that LNCs are stable in mucus, do not change mucus rheological properties, and improve Ptx diffusion at low concentrations, thus making these systems good candidates for Ptx oral delivery. The study of the physicochemical interaction between the LNC surface and its diffusion in mucus is now envisioned.

Femto-second laser beam with a low power density achieved a two-photon photodynamic cancer therapy with quantum dots.

Focusing the femto-second (fs) laser beam on the target was the usual way to carry out a two-photon excitation (TPE) in previous photodynamic therapy (PDT) studies. However, focusing the laser deep inside the tissues of the tumor is unrealistic due to tissue scattering, so that this focusing manner seems unfit for practical TPE PDT applications. In this work, we prepared a conjugate of quantum dots (QDs) and sulfonated aluminum phthalocyanine (AlPcS) for TPE PDT, because QDs have a very high two-photon absorption cross section (TPACS) and thus QDs can be excited by an unfocused 800 nm fs laser beam with a low power density and then transfer the energy to a conjugated AlPcS via fluorescence resonance energy transfer (FRET). The FRET efficiency of the QD-AlPcS conjugate in water was as high as 90%, and the FRET process of the cellular QD-AlPcS was also observed in both KB and HeLa cells under TPE of a 800 nm fs laser. The singlet oxygen (1O2) products were produced by the QD-AlPcS under the TPE of the unfocused 800 nm fs laser via FRET mediated PDT. Moreover, the QD-AlPcS can effectively destroy these cancer cells under the irradiation of the 800 nm unfocused fs laser beam with a power density of 92 mW mm-2, and particularly the killing efficiency of the TPE is comparable to that of the commonly used one-photon excitation (OPE) at visible wavelengths. These results highlight the potential of QD-AlPcS for TPE PDT with a near infrared wavelength.

Single-molecule photon stamping FRET spectroscopy study of enzymatic conformational dynamics.

The fluorescence resonant energy transfer (FRET) from a donor to an acceptor via transition dipole-dipole interactions decreases the donor's fluorescent lifetime. The donor's fluorescent lifetime decreases as the FRET efficiency increases, following the equation: E(FRET) = 1 - τ(DA)/τ(D), where τ(D) and τ(DA) are the donor fluorescence lifetime without FRET and with FRET. Accordingly, the FRET time trajectories associated with single-molecule conformational dynamics can be recorded by measuring the donor's lifetime fluctuations. In this article, we report our work on the use of a Cy3/Cy5-labeled enzyme, HPPK to demonstrate probing single-molecule conformational dynamics in an enzymatic reaction by measuring single-molecule FRET donor lifetime time trajectories. Compared with single-molecule fluorescence intensity-based FRET measurements, single-molecule lifetime-based FRET measurements are independent of fluorescence intensity. The latter has an advantage in terms of eliminating the analysis background noise from the acceptor fluorescence detection leak through noise, excitation light intensity noise, or light scattering noise due to local environmental factors, for example, in a AFM-tip correlated single-molecule FRET measurements. Furthermore, lifetime-based FRET also supports simultaneous single-molecule fluorescence anisotropy.

Optimization of FRET Microscopy for Live-Cell Imaging of Multiple Protein-Protein Interactions

Recent advances in fluorescence resonance energy transfer (FRET) microscopy have made it possible to measure simultaneously, multiple protein-protein interactions inside living cells. To further these methods, we integrated an improved multi-spectral imaging analysis method (N-way FRET) with optimized fluorophores for live-cell FRET microscopy. For 3-way FRET we determined the best cyan, yellow, and red (C-Y-R) FRET-trio by comparing the acceptor photobleaching FRET efficiencies of structurally similar linked fluorescent protein constructs. The pool of molecules examined was constrained to the available C, Y, R molecules with the best photophysical properties (CyPet/TFP1, mCitrine/YPet and TagRFP/mCherry, respectively). From this set, we determined that TFP, YPet or mCitrine and mCherry were the optimal FRET trio. For increasingly complex pathways, more fluorescent probes may be required. To extend this method to the analysis of 4-way FRET we examined fluorophore pairs with highly overlapping excitation and emission spectra (e.g. TFP and GFP or Citrine and GFP). These improvements should enhance the spatiotemporal resolution, the number of possible interacting probes and the reliability of FRET experiments leading to improved monitoring of multiple protein-protein interactions in complex pathways.

FRET-based Mapping of Regions involved in FKBP12.6 Binding to the Type 1 Ryanodine Receptor

The type 1 ryanodine receptor (RyR1) is an intracellular calcium channel that plays an integral role in skeletal muscle excitation-contraction coupling. The functional properties of this protein are regulated by numerous associated proteins, including FKBP12, a skeletal muscle protein that stabilizes full RyR1 channel gating. While the binding site of FKBP12 was originally identified at position 2458 of RyR1, subsequent reports have localized additional binding determinants within the N-terminal domain of RyR1. In this study, we used fluorescence resonance energy transfer (FRET) to determine the orientation and proximity of FKBP relative to sites either within the N-terminal domain or near amino acid position 2458. We utilized the cardiac muscle isoform FKBP12.6 (which binds to RyR1 at the same site as FKBP12 but with higher affinity and stability) labeled with the FRET donor Alexa Fluor 488 at either position 14, 44, 49, or 85. Specific binding of each labeled FKBP to RyR was confirmed in permeabilized HEK-293T cells expressing wtRyR1. Weak energy transfer was observed from FKBP12.6 to the FRET acceptor Cy3NTA, which was targeted to poly-histidine “tags” placed in the N-terminal domain. This result suggested that the acceptors were not proximal (∼90A away) to FKBP12.6 bound to RyR1. Next, we measured FRET from FKBP12.6 to Cy3NTA targeted to positions 2157, 2341, 2502 and 2777 of RyR1. FKBP12.6 binding to constructs containing His-tags at positions 2157 and 2502 was significantly reduced relative to binding to wtRyR1. In contrast, we detected strong and weak FRET efficiencies from FKBP12.6 to positions 2341 and 2777, respectively. These data suggest that FKBP12.6 binds near amino acid position 2341 but far from the N-terminal domain of RyR1. Supported by NIH grant R01AR059124 (to JDF and TG) and R01HL092097 (to RLC).

A gold nanorods-based fluorescent biosensor for the detection of hepatitis B virus DNA based on fluorescence resonance energy transfer

In this study, we designed a fluorescence resonance energy transfer system containing gold nanorods (AuNRs) and fluorescein (FAM) for the detection of hepatitis B virus DNA sequences. AuNRs were synthesized according to the seed-mediated surfactant-directed approach, and the surface of the AuNRs was wrapped with a thin layer of cetyltrimethylammonium bromide (CTAB), resulting in the AuNRs being positively charged. When FAM-tagged single-stranded DNA (FAM-ssDNA) was added into the AuNRs suspension, it was adsorbed onto the surface of the positively charged AuNRs and formed a FAM-ssDNA-CTAB-AuNRs ternary complex, the resulting structure led to a fluorescence resonance energy transfer (FRET) process from FAM to AuNRs and the fluorescence intensity of FAM was consequently quenched. When complementary target DNA was added to the FAM-ssDNA-CTAB-AuNRs complex solution, a further decrease in fluorescence intensity was observed because of an increased FRET efficiency. Under optimal conditions, the decline of the fluorescence intensity of FAM (ΔF) was linear with the concentration of the complementary DNA from 0.045 to 6.0 nmol L(-1) and the detection limit was as low as 15 pmol L(-1) (signal/noise ratio of 3). When this fluorescent DNA sensor was used to detect the polymerase chain reaction product of hepatitis B virus gene extracted from a positive real sample, a positive response was obtained. Impressively, the biosensor exhibits good selectivity, even for single-mismatched DNA detection.

One-pot fabrication of polymer nanoparticle-based chemosensors for Cu2+ detection in aqueous media

Cupric pollution is a global problem, and the development of stable and sensitive fluorescent probes for cupric ions in the water phase has long been sought. In the present study, we report on the fabrication of core–shell nanoparticle-based fluorescent chemosensors for Cu2+ detection in aqueous media. The core–shell nanoparticle sensor was prepared by a facile one-pot miniemulsion polymerization, in which the fluorescent dye (4-methamino-9-allyl-1,8-naphthalimide, MANI) was covalently incorporated into particle core and the Cu2+ ligand i.e. Vinylbenzylcyclam (VBC), chemically linked onto the surface. The cyclam-functionalized fluorescent polymeric nanoparticles exhibit a high affinity for Cu2+ ions in aqueous media. Upon the addition of Cu2+, the fluorescent emission of the MANI dye in nanoparticles can be quenched on the basis of intraparticle fluorescence resonance energy transfer (FRET) from the dye in the hydrophobic PMMA core to the Cu2+–cyclam complexes on the nanoparticle surface, and the nanoparticle sensor can selectively detect the Cu2+ in water with the detection limit of 500 nM. The observed FRET efficiencies (31.6–73.4%), as well as the distance (r) between MANI (donor) and Cu2+–cyclam complexes (acceptor), were also determined. No interference was observed from other metal ions, making it a highly sensitive and selective Cu2+ probe. Moreover, the nanoparticle-based fluorescent sensor was applicable in a relatively wide pH range (pH 4–10) in water and it exhibited excellent long-term photostability for Cu2+ detection (>45 days) in aqueous media; thus, this approach may provide a new strategy for ratiometric detection of analytes in environmental and biological applications.

Activated phosphonated trifunctional chelates for highly sensitive lanthanide-based FRET immunoassays applied to total prostate specific antigen detection

The first example of an activated phosphonated trifunctional chelate (TFC) is presented, which combines a non-macrocyclic coordination site for lanthanide coordination based on two aminobis-methylphosphonate coordinating arms, a central bispyrazolylpyridyl antenna and an N-hydroxysuccinimide ester in para position of the central pyridine as an activated function for the labeling of biomaterial. The synthesis of the TFC is presented together with photo-physical studies of the related Tb and Eu complexes. Excited state lifetime measurements in H2O and D2O confirmed an excellent shielding of the cation from water molecules with a hydration number of zero. The Tb complex provides a high photoluminescence (PL) quantum yield of 24% in aqueous solutions (0.01 M Tris-HCl, pH 7.4) and a very long luminescence lifetime of 2.6 ms. The activated ligand was conjugated to different biological compounds such as streptavidin, and a monoclonal antibody against total prostate specific antigen (TPSA). In combination with AlexaFluor647 (AF647) and crosslinked allophycocyanin (XL665) antibody (ABs) conjugates, homogeneous time-resolved Fluorescence Resonance Energy Transfer (FRET) immunoassays of TPSA were performed in serum samples. The Tb donor-dye acceptor FRET pairs provided large Förster distances of 5.3 nm (AF647) and 7.1 nm (XL665). A detailed time-resolved FRET analysis of Tb donor and dye acceptor PL decays revealed average donor-acceptor distances of 4.2 nm (AF647) and 6.3 nm (XL665) within the sandwich immunocomplex and FRET efficiencies of 0.79 and 0.68, respectively. Very low detection limits of 1.4 ng mL(-1) (43 pM) and 2.4 ng mL(-1) (74 pM) TPSA were determined using a KRYPTOR fluorescence immunoanalyzer. These results demonstrate the applicability of our novel Tb-bioconjugates for highly sensitive clinical diagnostics.

Direct molecular interaction of caveolin-3 with KCa1.1 channel in living HEK293 cell expression system

Caveolin family is supposed to be essential molecules for the formation of not only caveola structure on cell membrane but also functional molecular complexes in them with direct and/or indirect interaction with other membrane and/or submembrane associated proteins. The direct coupling of caveolin-1 (cav1) with large conductance Ca2+-activated K+ channel, KCa1.1 has been established in several types of cells and in expression system as well. The possible interaction of caveolin-3 (cav3), which shows expression in some differential tissues from cav1, with KCa1.1 remains to be determined. In the present study, the density of KCa1.1 current expressed in HEK293 cells was significantly reduced by the co-expression of cav3, as well as cav1. The co-localization and direct interaction of GFP- or CFP-labeled cav3 (GFP/CFP-cav3) with YFP- or mCherry-labeled KCa1.1 (KCa1.1-YFP/mCherry) were clearly demonstrated by single molecular image analyses using total internal reflection fluorescence (TIRF) microscopy and fluorescence resonance energy transfer (FRET) analyses with acceptor photobleaching method. The deletion of suggested cav1-binding motif in C terminus region of KCa1.1 (KCa1.1ΔCB-YFP) resulted in the marked decrease in cell surface expression, co-localization and FRET efficiency with CFP-cav3 and CFP-cav1. The FLAG-KCa1.1 co-immunoprecipitation with GFP-cav3 or GFP-cav1 also supported their direct molecular interaction. These results strongly suggest that cav3 possesses direct interaction with KCa1.1, presumably at the same domain for cav1 binding. This interaction regulates KCa1.1 expression to cell surface and the formation of functional molecular complex in caveolae in living cells.

Single Quantum Dot Based Nanosensor for Renin Assay

Evaluation of plasma renin activity is essential to the assessment of renin-related diseases such as hypertension, congestive heart failure, and cancers. Here, we develop a single quantum dot (QD) based nanosensor for sensitive detection of renin activity. This single-QD-based nanosensor consists of a streptavidin-coated QD and multiple biotinylated and Cy5-labeled peptide substrates, which form a QD/substrate/Cy5 complex where fluorescence resonance energy transfer (FRET) occurs with the QD as the donor and Cy5 as the acceptor. The presence of renin leads to the cleavage of the substrate and the separation of Cy5 from the QD and consequently the decrease of FRET efficiency and the reduction of Cy5 counts. Through the measurement of Cy5 counts by total internal reflection fluorescence (TIRF) microscopy, the renin activity can be quantitatively evaluated at the single-molecule level. This single-QD-based nanosensor can measure not only the renin concentration, but also the enzymatic velocity and the Michaelis-Menten kinetic parameters, and has significant advantages of simplicity, low cost with minimum sample consumption, and high sensitivity with a detection limit of 25 pM. This single-QD-based nanosensor might be further applied to monitor a variety of important enzymatic biomarkers such as kinases and endonuleases.

Conformational Dynamics of Titin PEVK Explored with FRET Spectroscopy

The proline-, glutamate-, valine-, and lysine-rich (PEVK) domain of the giant muscle protein titin is thought to be an intrinsically unstructured random-coil segment. Various observations suggest, however, that the domain may not be completely devoid of internal interactions and structural features. To test the validity of random polymer models for PEVK, we determined the mean end-to-end distances of an 11- and a 21-residue synthetic PEVK peptide, calculated from the efficiency of the fluorescence resonance energy transfer (FRET) between an N-terminal intrinsic tryptophan donor and a synthetically added C-terminal IAEDANS acceptor obtained in steady-state and time-resolved experiments. We find that the contour-length scaling of mean end-to-end distance deviates from predictions of a purely statistical polymer chain. Furthermore, the addition of guanidine hydrochloride decreased, whereas the addition of salt increased the FRET efficiency, pointing at the disruption of structure-stabilizing interactions. Increasing temperature between 10 and 50°C increased the normalized FRET efficiency in both peptides but with different trajectories, indicating that their elasticity and conformational stability are different. Simulations suggest that whereas the short PEVK peptide displays an overall random structure, the long PEVK peptide retains residual, loose helical configurations. Transitions in the local structure and dynamics of the PEVK domain may play a role in the modulation of passive muscle mechanics.

Partial Acceptor Photobleaching-Based Quantitative FRET Method Completely Overcoming Emission Spectral Crosstalks

Based on the quantitative fluorescence resonance energy transfer (FRET) method named PbFRET we reported recently, we herein developed a partial acceptor photobleaching-based quantitative FRET algorithm named B-PbFRET method. B-PbFRET overcomes not only the acceptor excitation crosstalk and donor emission spectral crosstalk but also the acceptor emission spectral crosstalk that harasses previous methods including fluorescence lifetime (FLIM), fluorescence recovery of donor after acceptor photobleaching, and acceptor sensitized emission (SE)-based methods. B-PbFRET method is implemented by simultaneously measuring the fluorescence intensity of both donor and acceptor channels at donor excitation before and after partial acceptor photobleaching, and it can directly measure the FRET efficiency (E) without any verified references. Based on the theoretical analysis of B-PbFRET, we also developed a more straightforward correction method named C-PbFRET to obtain the absolute E from the value measured by PbFRET for a given donor-acceptor pair. We validated both B-PbFRET and C-PbFRET methods by measuring the E of two linked constructs, 18AA and SCAT3 proteins, in single living cells, and our data demonstrated that both B-PbFRET and C-PbFRET methods can directly measure the absolute E of the linked constructs inside living cells under different degrees of acceptor emission spectral crosstalk.

Novel Adenosine Detection Method Based on Cationic Conjugated Polymer and Aptamer

We designed a novel adenosine detection system based on cationic conjugated polymer(CCP) and aptamer.A fluorescein-modified short-chain DNA can partially hybridize with adenosine aptamer to form a double stand DNA(dsDNA).The CCP associated with the dsDNA by electrostatic attractions and an efficient fluorescence resonance energy transfer(FRET) from the CCP to the fluorescein occurred.When adenosine was added,it bonded specifically to the aptamer,making the dsDNA break into single stand DNA(ssDNA),then the FRET efficiency decreased.Thus,we could detect the adenosine quickly and easily through the high signal discrimination between dsDNA and ssDNA with the CCP.The results show that the degree of FRET efficiency decrease is related to the concentration of adenosine.This detection method has the advantages including easy operations,low costs,and relatively high sensitivity;moreover,it can be extended to detections of other targets owning aptamers.

Antidepressant drugs promote the heterodimerization of the dopamine D2 and somatostatin Sst5 receptors – fluorescence in vitro studies

BackgroundThe interaction between the dopaminergic and somatostatinergic systems and their role in mood regulation have been well-documented. Therefore, we decided to investigate the effect of antidepressant drugs on the heterodimerization of the dopamine D2 and somatostatin Sst5 receptors. MethodsThe human receptor proteins were tagged with fluorescent proteins, expressed in the HEK 293 cells and incubated with antidepressant drugs: desipramine and citalopram. To determine the FRET efficiency, the fluorescence resonance energy transfer (FRET) and photobleaching confocal microscopy techniques were used. ResultsWe found that the efficiency of FRET is markedly increased in cells coexpressing the somatostatin Sst5 and dopamine D2 receptors after 48h of incubation with desipramine and citalopram. ConclusionsIn the present study we provide physical evidence, based on FRET analysis, that antidepressants increase Sst5 and D2 receptors heterodimerization. The effect is specific because desipramine in the incubation medium uncouples other pairs of receptors, such as the dopamine D1-D2 receptors.

Synthesis and Characterization of Novel Pyrene-Dendronized Porphyrins Exhibiting Efficient Fluorescence Resonance Energy Transfer: Optical and Photophysical Properties

A novel series of pyrene dendronized porphyrins bearing two and four pyrenyl groups (Py(2)-TMEG1 and Py(4)-TMEG2) were successfully synthesized. First and second generation Fréchet type dendrons (Py(2)-G1OH and Py(4)-G2OH) were prepared from 1-pyrenylbutanol and 3,5-dihydroxybenzyl alcohol. These compounds were further linked to a trimesitylphenylporphyrin containing a butyric acid spacer via an esterification reaction to obtain the desired products. Dendrons and dendronized porphyrins were fully characterized by FTIR and (1)H NMR spectroscopy and their molecular weights were determined by matrix-assisted laser desorption ionization time of flight mass spectrometry. Their optical and photophysical properties were studied by absorption and fluorescence spectroscopies. The formation of dynamic excimers was detected in the pyrene-labeled dendrons, with more excimer being produced in the higher generation dendron. The fluorescence spectra of the pyrene dendronized porphyrins exhibited a significant decrease in the amount of pyrene monomer and excimer emission, jointly with the appearance of a new emission band at 661 nm characteristic of porphyrin emission, an indication that fluorescence resonance energy transfer (FRET) occurred from one of the excited pyrene species to the porphyrin. The FRET efficiency was found to be almost quantitative ranging between 97% and 99% depending on the construct. Model Free analysis of the fluorescence decays acquired with the pyrene monomer, excimer, and porphyrin core established that only residual pyrene excimer formation in the dendrons could occur before FRET from the excited pyrene monomer to the ground-state porphyrin core.

Investigating Ligand–Receptor Interactions at Bilayer Surface Using Electronic Absorption Spectroscopy and Fluorescence Resonance Energy Transfer

We investigate interactions between receptors and ligands at bilayer surface of polydiacetylene (PDA) liposomal nanoparticles using changes in electronic absorption spectroscopy and fluorescence resonance energy transfer (FRET). We study the effect of mode of linkage (covalent versus noncovalent) between the receptor and liposome bilayer. We also examine the effect of size-dependent interactions between liposome and analyte through electronic absorption and FRET responses. Glucose (receptor) molecules were either covalently or noncovalently attached at the bilayer of nanoparticles, and they provided selectivity for molecular interactions between glucose and glycoprotein ligands of E. coli. These interactions induced stress on conjugated PDA chain which resulted in changes (blue to red) in the absorption spectrum of PDA. The changes in electronic absorbance also led to changes in FRET efficiency between conjugated PDA chains (acceptor) and fluorophores (Sulphorhodamine-101) (donor) attached to the bilayer surface. Interestingly, we did not find significant differences in UV-vis and FRET responses for covalently and noncovalently bound glucose to liposomes following their interactions with E. coli. We attributed these results to close proximity of glucose receptor molecules to the liposome bilayer surface such that induced stress were similar in both the cases. We also found that PDA emission from direct excitation mechanism was ~2-10 times larger than that of the FRET-based response. These differences in emission signals were attributed to three major reasons: nonspecific interactions between E. coli and liposomes, size differences between analyte and liposomes, and a much higher PDA concentration with respect to sulforhodamine (SR-101). We have proposed a model to explain our experimental observations. Our fundamental studies reported here will help in enhancing our knowledge regarding interactions involved between soft particles at molecular levels.

P4‐071: Involvement of X11/X11L in Alzheimer's‐related beta‐amyloid generation and epileptogenesis

Background: Amyloid beta (Ab), originating from cleavage of APP (amyloid precursor protein), is a hallmark of Alzheimer’s disease. P75NTR is known to regulate Abeta deposition in the brain but its role in APP trafficking and processing is not known. Methods: We use FRET (fluorescence resonance energy transfer) analysis to characterize the nature of the APPp75NTR interaction in HEK293 cells which were transfected with p75NTR-CFP and APP-YFP plasmids and controls. These cells were subjected to a range of neurotrophins, and Ab, at differing concentrations. Co-immunoprecipitation and fractionation were used to examine the interaction between p75NTR and APP. Results: The FRET efficiency of APP-p75NTR was significantly higher than the negative control. All neurotrophins triggered an increase in the FRETefficiency between p75NTR and APP. Interestingly, Ab at all concentrations triggered a dramatic increase in FRET efficiency.The interaction increased dramatically within the first 1-3 minutes of the addition of Ab to the cells. Co-IP data support that Abeta enhanced the interaction between APP and p75NTR. When a phosphorylation inhibitor KT5720 was added to transfected cells with added Ab, FRET efficiency dropped. Reciprocally, when a phosphorylation activator 8-BrcAMP was added, FRET efficiency increased significantly. This data seems to support the idea that p75NTR-APP interaction is under the influence of a phosphorylation event. Furthermore FRET efficiency dropped further by blocking endogenous Ab with an antibody 6E10, indicating endogenous Ab may increase the interaction of APP and p75NTR. These results could suggest that Ab may have a positive feed forward mechanism that increases an output of Ab via the interaction among Abeta, p75NTR and APP. Conclusions: We conclude that p75NTR ligands Ab and neurotrophins enhance the interaction of p75NTR-APP in a phosphorylation dependent manner, and the increased interaction of p75NTR-APPmay participate in the pathogenesis of AD.

QUANTITATIVE FRET MEASUREMENT BASED ON CONFOCAL MICROSCOPY IMAGING AND PARTIAL ACCEPTOR PHOTOBLEACHING

Fluorescence resonance energy transfer (FRET) technology had been widely used to study protein–protein interactions in living cells. In this study, we developed a ROI-PbFRET method to real-time quantitate the FRET efficiency of FRET construct in living cells by combining the region of interest (ROI) function of confocal microscope and partial acceptor photobleaching. We validated the ROI-PbFRET method using GFPs-based FRET constructs including 18AA and SCAT3, and used it to quantitatively monitor the dynamics of caspase-3 activation in single live cells stably expressing SCAT3 during staurosporine (STS)-induced apoptosis. Our results for the first demonstrate that ROI-PbFRET method is a powerful potential tool for detecting the dynamics of molecular interactions in live cells.