Intramolecular Fluorescence Resonance Energy Transfer Research Articles

Dynamics-Driven Allostery Underlies Ca2+-Mediated Release of SERCA Inhibition by Phospholamban

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential for intracellular Ca2+ transport in myocytes. Ca2+-dependent activation of SERCA-PLB provides a control function that regulates cytosolic and SR Ca2+ levels. Although experimental and computational studies alone have led to a greater insight into SERCA-PLB regulation, the structural mechanisms for Ca2+ binding reversing inhibition of the complex remain poorly understood. Therefore, we have performed atomistic simulations totaling 32.7 μs and cell-based intramolecular fluorescence resonance energy transfer (FRET) experiments to determine structural changes of PLB-bound SERCA in response to binding of a single Ca2+ ion. Complementary MD simulations and FRET experiments showed that open-to-closed transitions in the structure of the headpiece underlie PLB inhibition of SERCA, and binding of a single Ca2+ ion is sufficient to shift the protein population toward a structurally closed structure of the complex. Closure is accompanied by functional interactions between the N-domain β5–β6 loop and the A-domain and the displacement of the catalytic phosphorylation domain toward a competent structure. We propose that reversal of SERCA-PLB inhibition is achieved by stringing together its controlling modules (A-domain and loop Nβ5–β6) with catalytic elements (P-domain) to regulate function during intracellular Ca2+ signaling. We conclude that binding of a single Ca2+ is a critical mediator of allosteric signaling that dictates structural changes and motions that relieve SERCA inhibition by PLB. Understanding allosteric regulation is of paramount importance to guide therapeutic modulation of SERCA and other evolutionarily related ion-motive ATPases.

PH-responsive intramolecular FRET-based self-tracking polymer prodrug nanoparticles for real-time tumor intracellular drug release monitoring and imaging.

An intramolecular fluorescence resonance energy transfer (FRET)-based macromolecular theranostic prodrug was designed by directly conjugating Doxorubicin (DOX) as the FRET acceptor onto the naphthalimide side groups in the fluorescent copolymer PPEGMA20-PNAP8 as the FRET energy donor via an acid-labile imine bond, without a fluorogenic linker. The proposed PPEGMA20-PNAP8-DOX theranostic prodrug showed a high DOX content of 24.3% owing to a conjugation efficiency of>93% under mild conjugation conditions. It could easily self-assemble into unique theranostic nanoparticles with a Dh of 71nm. The theranostic nanoparticles showed excellent pH-triggered DOX release performance with very low premature drug leakage of 6.3% in normal physiological medium over 129h, while>91% of the conjugated DOX was released in the acidic tumor intracellular microenvironment. MTT assays indicated the enhanced antitumor efficacy of the proposed theranostic nanoparticles compared with free DOX. Furthermore, because drug release was triggered by pH, orange fluorescence was restored to the blue fluorescence of the backbone copolymer. Such self-tracking pH-responsive colorful fluorescence variations during intracellular drug delivery and release are expected to allow real-time tumor intracellular drug release monitoring and imaging diagnosis.

Amphiphilic Fluorescence Resonance Energy-Transfer Dyes: Synthesis, Fluorescence, and Aggregation Behavior in Water.

Amphiphilic pyrene/perylene bis-chromophore dyes were synthesized from unsymmetrically substituted perylene bisimide dyes, which were obtained through three synthetic methods. The optical and aggregation behaviors of these functional dyes were studied by means of UV/Vis absorption and fluorescence spectroscopy, dynamic light scattering, and TEM. These dyes are highly fluorescent and cover the whole visible-light region. A donor/acceptor dye displays intramolecular fluorescence resonance energy transfer (FRET), with a high efficiency of up to 96.4 % from pyrene to perylene bisimide chromophores, which leads to a high fluorescence color sensitivity to environmental polarity. Under a λ=365 nm UV lamp, the light-emitting colors of the donor/acceptor dye change from green to yellow with increasing solvent polarity, which demonstrates application potential as a new class of FERT probes. The donor/acceptor dye in water was assembled into hollow vesicles with a narrow size distribution. The bilayer structure of the vesicular wall was directly observed by means of TEM. These vesicular aggregates in water are fluorescent at λ=650-850 nm within the near-infrared region.

Reversible FRET Fluorescent Probe for Ratiometric Tracking of Endogenous Fe3+ in Ferroptosis.

A reversible fluorescent probe DRhFe is devised by linking spirolactam rhodamine and dansylamide through an Fe3+ ionophore N2-hydroxyethyldiethylenetriamine, where Fe3+ chelation generating rhodamine B can switch intramolecular fluorescence resonance energy transfer for ratiometric Fe3+ sensing. Probe DRhFe exhibits an Fe3+-specific emission shift from 483 to 576 nm, and this ratiometric response has been successfully applied to monitor labile Fe3+ fluctuations in cells undergoing ferroptosis.

A glutathione-responsive photosensitizer with fluorescence resonance energy transfer characteristics for imaging-guided targeting photodynamic therapy

Here, we have synthesized and characterized a novel activatable photosensitizer (PS) 8a in which two well-designed boron dipyrromethene (BODIPY) derivatives are utilized as the photosensitizing fluorophore and quencher respectively, which are connected by a disulfide linker via two successive Cu (І) catalyzed click reactions. The fluorescence emission and singlet oxygen production of 8a are suppressed via intramolecular fluorescence resonance energy transfer (FRET) from the excited BODIPY-based PS part to quencher unit, but both of them can be simultaneously switched on by cancer-related biothiol glutathione (GSH) in phosphate buffered saline (PBS) solution with 0.05% Tween 80 as a result of cleavage of disulfide. Also, 8a exhibits a bright fluorescence image and a substantial ROS production in A549 human lung adenocarcinoma, HeLa human cervical carcinoma and H22 mouse hepatoma cells having a relatively high concentration of GSH, thereby leading to a significant photocytotoxicity, with IC50 values as low as 0.44 μM, 0.67 μM and 0.48 μM, respectively. In addition, the photosensitizer can be effectively activated and imaged in H22 transplanted hepatoma tumors of mice and shows a strong inhibition on tumor growth. All these results suggest that such a GSH-responsive photosensitizer based on FRET mechanism may provide a new strategy for tumor-targeted and fluorescence imaging-guided cancer therapy.

Synthesis, characterization, photophysical and intramolecular energy transfer properties of oxy-naphthylchalcone appended cyclotriphosphazene cores

In this study, three novel oxy-naphthylchalcone appended cyclotriphosphazene derivatives (3, 4 and 5) were obtained by the nucleophilic substitution reactions of cyclotriphosphazene cores (1, 1a and 1b) with (E)-3-(4-hydroxyphenyl)-1-(naphthalene-2-yl) prop-2-en-1-one (2) in presence of K2CO3/acetone. Novel compounds 3, 4 and 5 were characterized with using NMR (1H and 31P), elemental analysis, mass and FT-IR spectroscopies. In addition, molecular and crystal structures of 2 and 3 were determined by single crystal X-ray crystallography. The photophysical and intramolecular energy transfer properties of novel oxy-naphthylchalcone appended cyclotriphosphazene derivatives (3, 4 and 5) and their precursors (1a and 1b) were examined by steady state-/time resolved fluorescence spectroscopy, UV–Vis absorption spectroscopy and excitation-emission matrix (EEM) analysis. According to obtained results, novel target compounds (4 and 5) demonstrate a highly efficient intramolecular fluorescence resonance energy transfer (FRET) between excited phenol/naphthol groups and oxy-naphthylchalcone group.

Restoration of MARCK enhances chemosensitivity in cancer

PurposeIncreased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1.MethodsCRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU).ResultsWe found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU.ConclusionsCells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.

A phthalocyanine-porphyrin triad for ratiometric fluorescent detection of Lead(II) ions

A phthalocyanine-porphyrin hetero-triad H2Pc-β-(ZnPor)2 (1) was designed for the ratiometric fluorescent detection of lead(II) ions. The triad 1 features a high efficient intramolecular fluorescence resonance energy transfer (FRET) process from the two zinc-porphyrin (ZnPor) units to the metal-free phthalocyanine (H2Pc) unit. The selective binding of Pb2+ to H2Pc can effectively quench the emission of phthalocyanine unit, also recover the emission of ZnPor units by suppressing the intramolecular FRET process, thus leading to remarkable ratiometric fluorescent responses. The emission intensity ratio of ZnPor and H2Pc (F605/F700) experiences an 82-fold enhancement upon the addition of Pb2+ (0–3 equiv) to a solution of 1 (2.0 μM in THF/CH3OH, 4:1 in v/v), and displays a good linear relationship to Pb2+ concentration in the range of 0–2.0 μM, resulting in a limit of detection (LOD) value down to 4.1 nM (0.86 ppb).

Monitoring of dynamic ATP level changes by oligomycin-modulated ATP synthase inhibition in SW480 cancer cells using fluorescent \u201cOn-Off\u201d switching DNA aptamer

Adenosine triphosphate (ATP) is the main energy source in cells and an important biomolecule participating in cellular reactions in living organisms. Since the ATP level changes dynamically reflecting the development of a debilitating disease or carcinogenesis, we have focused in this work on monitoring of the oligomycin (OMC)-modulated ATP synthase inhibition using a fluorescent-switching DNA aptamer designed for the detection of ATP (Apt(ATP)), as the model for studies of dynamic ATP level variation. The behavior of the ATP aptamer has been characterized using fluorescence spectroscopy. The Intramolecular fluorescence resonance energy transfer (iFRET) operates in the proposed aptamer from the FAM dye moiety to guanines of the aptamer G-quadruplex when the target ATP is present and binds to the aptamer changing its conformation. The iFRET process enables the detection of ATP down to the limit of detection, LOD = 17 μM, without resorting to any extra chemi-amplification schemes. The selectivity coefficients for relevant interferent triphosphates (UTP, GTP, and CTP) are low for the same concentration as that of ATP. We have demonstrated an efficient transfection of intact cells and OMC-treated SW480 colon cancer cells with Apt(ATP), using microscopic imaging, iFRET measurements, and cell viability testing with MTT method. The applicability of the switching DNA aptamer for the analysis of real samples, obtained by lysis of SW480 cells, was also tested. The proposed Apt(ATP) may be considered as a viable candidate for utilization in measurements of dynamic ATP level modulation in cells in different stages of cancer development and testing of new drugs in pharmacological studies.Graphical abstract

Temperature-Dependent Expression of a CFP-YFP FRET Diacylglycerol Sensor Enables Multiple-Read Screening for Compounds That Affect C1 Domains

Intramolecular CFP-YFP fluorescence resonance energy transfer (FRET) sensors expressed in cells are powerful research tools but have seen relatively little use in screening. We exploited the discovery that the expression of a CFP-YFP FRET diacylglycerol sensor (DAGR) increases over time when cells are incubated at room temperature to assess requirements for robust measurements using a Molecular Devices Spectramax i3x fluorescence plate reader. Expression levels resulting in YFP fluorescence >10-fold higher than untransfected cells and phorbol ester-stimulated FRET ratio changes of 60% or more were required to consistently give robust Z′ > 0.5. As a means of confirming that these conditions are suitable for screening, we developed a novel multiple-read protocol to assay the NCI’s Mechanistic Set III for agonists and antagonists of C1 domain activation. Sixteen compounds prevented C1 domain translocation. However, none blocked phorbol ester-stimulated protein kinase C (PKC) activity assessed using a phospho-specific antibody—six actually stimulated PKC activity. Cytometry, which produces higher Z′ for a given FRET ratio change, might have been a better approach for discovering antagonists, as it would have allowed lower phorbol ester concentrations to be used. We conclude that CFP-YFP FRET measured in a Spectramax i3x plate reader can be used for screening under the conditions we defined. Our strategy of varying expression level and FRET ratio could be useful to others for determining conditions needed for robust cell-based intramolecular CFP-YFP FRET measurements on their instrumentation.

A FRET method for investigating dimer/monomer status and conformation of the UVR8 photoreceptor

The photoreceptor UVR8 has a pivotal role in mediating plant responses to UV-B wavelengths. Dimeric UVR8 dissociates into monomers following UV-B photoreception, and there is evidence that this process is accompanied by conformational changes that may facilitate interaction of UVR8 with other proteins to initiate signaling. Hence monitoring UVR8 dimer/monomer status and conformation is key to understanding UVR8 action. Here we have used Fluorescence Resonance Energy Transfer (FRET) to study these processes in both wild-type and mutant UVR8 proteins in vivo. UVR8 was fused to GFP and mCherry at the C- and N-termini, respectively and both the FRET efficiency and loss of GFP fluorescence after photo-bleaching were measured. In addition, measurements were made for UVR8 fused to either GFP or mCherry to eliminate intra-molecular FRET signals. The results indicate that dissociation of UVR8 dimer to monomer principally accounts for the loss of FRET signal for wild-type UVR8 and there is little evidence of a contribution from conformational change in vivo. Examination of plants expressing UVR8W285F and UVR8D96N,D107N are consistent with these mutant proteins being constitutively dimeric and mono-meric, respectively. The methods employed here will be valuable for monitoring UVR8 dimer/monomer status in vivo in relation to signaling, and will facilitate characterization of dimer/monomer status and conformation of further UVR8 mutants.

Live-Cell FRET Imaging Reveals a Role of ERK Activity Dynamics in Thymocyte Motility

Thymocyte motility is key to orchestrating migration between different thymic microenvironments at the appropriate developmental stage. Several lines of evidence suggest that extracellular signal-regulated kinase (ERK) signaling pathways plays critical roles in T cell development. Nevertheless, the dynamics of ERK activity and its role in regulating cell motility remain largely unknown due to technical difficulties. Therefore, there is an increasing demand for genetic reporter systems that provide the information of thymocyte motility and ERK signaling under physiological condition in real time.To meet the demand, we developed transgenic mice expressing fluorescence resonance energy transfer (FRET) based biosensor for ERK incorporated into the ROSA26 locus conditionally. EKAREV is a genetically encoded intramolecular FRET biosensor for monitoring ERK activity in living cells. We introduced cDNAs of EKAREV into the ROSA26 locus to generate mouse lines named R26-EKAREV-NLSfl/+. In these mouse lines, EKAREV will be expressed in the nucleus after Cre-mediated excision of the loxP-flanked tdKeima-coding sequence. To study ERK activity dynamics in T cells, R26-EKAREV-NLSfl/+ were crossed with Lck-Cre mice to generate R26-EKAREV-NLSlck/+. R26-EKAREV-NLSlck/+ showed uniform and high-level expression of EKAREV in lymphocytes and allowed us to examine the ERK activity of T cells in vivo.After initial characterization of R26-EKAREV-NLSlck/+, we attempted to unravel the potential crosstalk between ERK activity dynamics and cell motility within the thymic microenvironment. Long-term in vivo imaging of thymus was difficult due to the anatomical location abutting the heart. To circumvent this problem, thymocytes obtained from R26-EKAREV-NLSlck/+ were overlaid on C57BL/6 (WT) thymic slices and observed under two-photon excitation microscopy. During the course of experiments, we noticed a negative correlation between ERK activity and migration speed of thymocytes. To confirm this observation, thymocytes were cultured in the presence or absence of an mitogen-activated protein kinase/ERK kinase (MEK) inhibitor prior to transfer onto thymic slices. Treatment of thymocytes with MEK inhibitor increased averaged migration speed in both double-positive (DP) and single-positive (SP) subsets. Moreover, time-lapse imaging of each subsets of thymocytes revealed that the deviation of ERK activity from the average of individual cells regulated cell motility in CD4-SP, while the absolute value of ERK activity regulated cell motility in DP and CD8-SP. Collectively, these results suggest that CD4-SP is unique in that the ERK activity dynamics negatively regulate cell motility.Which signal regulates ERK activity and cell motility of CD4-SP in the medulla? Interaction between T cell receptor (TCR) and major histocompatibility complex (MHC) is known to regulate ERK activity and cell motility of T cells, but their direct relationship remains unknown in tissues. To examine this possibility, CD4-SP thymocytes were overlaid onto WT or MHC class II knockout (KO) thymic slices. When overlaid onto KO thymic slices, the average migration speed was significantly increased, indicating that CD4-SP cells arrest upon TCR-MHC II interaction. Moreover, the variance of ERK activity, but not the average of it, in CD4-SP cells on KO thymic slices was decreased, suggesting that TCR-MHC II interaction contribute the variance of ERK activity in CD4-SP.Our findings unravel that the deviation of ERK activity induced by TCR-MHC interactions negatively regulate cell motility of CD4-SP in the medulla. The live-cell FRET imaging of ERK activity will open a new window to understand the dynamic nature and the diverse functions of ERK signaling in T cell biology. DisclosuresTakaori-Kondo:Janssen Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding.

Development of Novel Intramolecular FRET-Based ABC Transporter Biosensors to Identify New Substrates and Modulators.

Multidrug resistance protein 1 (MRP1) can efflux a wide variety of molecules including toxic chemicals, drugs, and their derivatives out of cells. Substrates of MRP1 include anti-cancer agents, antibiotics, anti-virals, anti-human immunodeficiency virus (HIV), and many other drugs. To identify novel substrates and modulators of MRP1 by exploiting intramolecular fluorescence resonance energy transfer (FRET), we genetically engineered six different two-color MRP1 proteins by changing green fluorescent protein (GFP) insertion sites, while keeping the red fluorescent protein (RFP) at the C-terminal of MRP1. Four of six recombinant proteins showed normal expression, localization, and transport activity. We quantified intramolecular FRET using ensemble fluorescence spectroscopy in response to binding of known substrate or ATP alone, substrate/ATP, and trapping of the transporter in closed conformation by vanadate. Recombinant MRP1 proteins GR-881, GR-888, and GR-905 exhibited reproducible and higher FRET changes under all tested conditions and are very promising for use as MRP1 biosensors. Furthermore, we used GR-881 to screen 40 novel anti-cancer drugs and identified 10 hits that potentially directly interact with MRP1 and could be substrates or modulators. Profiling of drug libraries for interaction with MRP1 can provide very useful information to improve the efficacy and reduce the toxicity of various therapies.

Redistribution of SERCA calcium pump conformers during intracellular calcium signaling

The conformational changes of a calcium transport ATPase were investigated with molecular dynamics (MD) simulations as well as fluorescence resonance energy transfer (FRET) measurements to determine the significance of a discrete structural element for regulation of the conformational dynamics of the transport cycle. Previous MD simulations indicated that a loop in the cytosolic domain of the SERCA calcium transporter facilitates an open-to-closed structural transition. To investigate the significance of this structural element, we performed additional MD simulations and new biophysical measurements of SERCA structure and function. Rationally designed in silico mutations of three acidic residues of the loop decreased SERCA domain-domain contacts and increased domain-domain separation distances. Principal component analysis of MD simulations suggested decreased sampling of compact conformations upon N-loop mutagenesis. Deficits in headpiece structural dynamics were also detected by measuring intramolecular FRET of a Cer-YFP-SERCA construct (2-color SERCA). Compared with WT, the mutated 2-color SERCA shows a partial FRET response to calcium, whereas retaining full responsiveness to the inhibitor thapsigargin. Functional measurements showed that the mutated transporter still hydrolyzes ATP and transports calcium, but that maximal enzyme activity is reduced while maintaining similar calcium affinity. In live cells, calcium elevations resulted in concomitant FRET changes as the population of WT 2-color SERCA molecules redistributed among intermediates of the transport cycle. Our results provide novel insights on how the population of SERCA pumps responds to dynamic changes in intracellular calcium.

A platform of BRET-FRET hybrid biosensors for optogenetics, chemical screening, and in vivo imaging

Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.

Visualization of the activation of the histamine H3 receptor (H3R) using novel fluorescence resonance energy transfer biosensors and their potential application to the study of H3R pharmacology.

Activation of the histamine-3 receptor (H3R) is involved in memory processes and cognitive action, while blocking H3R activation can slow the progression of neurological disorders, such as Alzheimer's disease, schizophrenia and narcolepsy. To date, however, no direct way to examine the activation of H3R has been utilized. Here, we describe a novel biosensor that can visualize the activation of H3R through an intramolecular fluorescence resonance energy transfer (FRET) signal. To achieve this, we constructed an intramolecular H3R FRET sensor with cyan fluorescent protein (CFP) attached at the C terminus and yellow fluorescent protein (YFP) inserted into the third intracellular loop. The sensor was found to internalize normally on agonist treatment. We measured FRET signals between the donor CFP and the acceptor YFP in living cells in real time, the results of which indicated that H3R agonist treatment (imetit or histamine) increases the FRET signal in a time- and concentration-dependent manner with Kon and Koff values consistent with published data and which maybe correlated with decreasing cAMP levels and the promotion of ERK1/2 phosphorylation. The FRET signal was inhibited by H3R antagonists, and the introduction of mutations at F419A, F423A, L426A and L427A, once again, the promotion of ERK1/2 phosphorylation, was diminished. Thus, we have built a H3R biosensor which can visualize the activation of receptor through real-time structure changes and which can obtain pharmacological kinetic data at the same time. The FRET signals may allow the sensor to become a useful tool for screening compounds and optimizing useful ligands.

Assessing the real-time activation of the cannabinoid CB1 receptor and the associated structural changes using a FRET biosensor.

The cannabinoid receptor 1 (CB1) is mainly expressed in the nervous system and regulates learning, memory processes, pain and energy metabolism. However, there is no way to directly measure its activation. In this study, we constructed a CB1 intramolecular fluorescence resonance energy transfer (FRET) sensor, which could measure CB1 activation by monitoring structural changes between the third intracellular loop and the C-terminal tail. CB1 agonists induced a time- and concentration-dependent increase in the FRET signal, corresponding to a reduction in the distance between the third intracellular loop and the C-terminal tail. This, in turn, mobilized intracellular Ca2+, inhibited cAMP accumulation, and increased phosphorylation of the ERK1/2 MAP kinases. The activation kinetics detected using this method were consistent with those from previous reports. Moreover, the increased FRET signal was markedly inhibited by the CB1 antagonist rimonabant, which also reduced phosphorylation of the ERK1/2 MAP kinases. We mutated a single cysteine residue in the sensor (at position 257 or 264) to alanine. Both mutation reduced the agonist-induced increase in FRET signal and structural changes in the CB1 receptor, which attenuated phosphorylation of the ERK1/2 MAP kinases. In summary, our sensor directly assesses the kinetics of CB1 activation in real-time and can be used to monitor CB1 structure and function.

The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET

Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

Reversible photoluminescence switching in supramolecular ultrathin films architecture

Host-guest systems have attracted increasing interest in designing stimuli-responsive devices in recent years. In this paper, a photo-switchable supramolecular ultrathin films (UTFs) with high on/off contrast and good reversibility has been constructed based on the host-guest assembly between cationic layered double hydroxide (LDH) nanosheets and anionic [Eu(SiW10MoO39)2]13− (denoted as EuSiWMo). The luminescence of the UTFs can be efficiently quenched under UV light (<320 nm) irradiation due to the intramolecular fluorescence resonance energy transfer (im-FRET) from the excitation states of Eu3+ to the intervalence charge transfer (IVCT) band of the photoreduced EuSiWMo, which can be efficiently suppressed by oxygen and the luminescence of the UTFs can therefore be recovered to the initial state, making the UTFs potentially available in fabricating optical memory devices and optical switches, etc.

Ratiometric Fluorescent Detection of Pb2+ by FRET-Based Phthalocyanine-Porphyrin Dyads.

Sensitive and selective detection of Pb2+ is a very worthwhile endeavor in terms of both human health and environmental protection, as the heavy metal is fairly ubiquitous and highly toxic. In this study, we designed phthalocyanine-porphyrin (Pc-Por) heterodyads, namely, H2Pc-α-ZnPor (1) and H2Pc-β-ZnPor (2), by connecting a zinc(II) porphyrin moiety to the nonperipheral (α) or peripheral (β) position of a metal-free phthalocyanine moiety. Upon excitation at the porphyrin Soret region (420 nm), both of the dyads exhibited not only a porphyrin emission (605 nm) but also a phthalocyanine emission (ca. 700 nm), indicating the occurrence of intramolecular fluorescence resonance energy transfer (FRET) processes from the porphyrin donor to the phthalocyanine acceptor. The dyads can selectively bind Pb2+ in the phthalocyanine core leading to a red shift of the phthalocyanine absorption and thus a decrease of spectral overlap between the porphyrin emission and phthalocyanine absorption, which in turn suppresses the intramolecular FRET. In addition, the binding of Pb2+ can highly quench the emission of phthalocyanine by heavy-metal ion effects. The synergistic coupled functions endow the dyads with remarkable ratiometric fluorescent responses at two distinct wavelengths (F605/F703 for 1 and F605/F700 for 2). The emission intensity ratio increased as a linear function to the concentration of Pb2+ in the range of 0-4.0 μM, whereas the detection limits were determined to be 3.4 × 10-9 and 2.2 × 10-8 M for 1 and 2, respectively. Furthermore, by comparative study of 1 and 2, the effects of distance and relative orientation between Pc and ZnPor fluorophores on the FRET efficiency and sensing performance were highlighted, which is helpful for further optimizing such FRET systems.