Strong Fluorescence Resonance Energy Transfer Research Articles

A fluorescence biosensor based on double-stranded DNA and a cationic conjugated polymer coupled with exonuclease III for acrylamide detection

As a toxic substance on human health produced in food thermal treatment, simple analytical approaches are highly desired for the detection of acrylamide (ACR) in foods. With the aid of exonuclease III (Exo III), a simple fluorescence sensor was proposed based on carboxyfluorescein-labeled double-stranded DNA (FAM-dsDNA) and a cationic conjugated polymer (PFP). Fluorescence resonance energy transfer (FRET) efficiency between FAM and PFP was changed with and without ACR. When ACR was present, ACR and single-stranded DNA (P1, ssDNA) formed an adduct, allowing free FAM-labeled complementarity strand DNA (P2, FAM-csDNA) to appear in the solution and avoiding the digestion of P2 by Exo III. After the addition of PFP, the interaction of PFP and FAM induced strong FRET. Under optimized conditions, ACR was detected with a limit of detection (LOD) of 0.16 μM. According to this biosensor, a LOD of 1.3 μM in water extract samples was observed with a good recovery rate (95–110 %).

The First FRET-Based RNA Aptamer NanoKit for Sensitively and Specifically Detecting c-di-GMP.

An effective method to identify c-di-GMP may significantly facilitate the exploration of its signaling pathways and bacterial pathogenesis. Herein, we have developed the first conjugated polymer-amplified RNA aptamer NanoKit with a unique core-shell-shell architecture, which combines the advantages of high selectivity of RNA aptamers and high sensitivity of strong fluorescence resonance energy transfer (FRET) effect, for precisely detecting c-di-GMP. We identified that NanoKit could selectively detect c-di-GMP with a low detection limit of 50 pM. Importantly, NanoKit could identify bacterial species and physiological states, such as planktonic, biofilm, and even antibiotic-resistance, on the basis of their different c-di-GMP expression patterns. Particularly, NanoKit could distinguish bacterial infection and inflammation and identify Pseudomonas aeruginosa associated pneumonia and sepsis, thereby guiding treatment choice and monitoring antibiotic effects. Therefore, NanoKit provides a promising strategy to rapidly identify c-di-GMP and its associated diseases and may benefit for pathophoresis management.

Leaf-inspired fluorescence-switchable nanosystem for visual loading of mixed drugs and targeted delivery

Inspired by the structure of leaves that possesses hollow veins and mesophyll with high surface area, a novel MnO2-based biomimetic fluorescence-switchable nanosystem was constructed to visually monitor drug loading and deliver mixed drugs. Leaf-like MnO2 with hollow tubes and wrinkled sheets was first synthesized, and further modified by dopamine for effective loading both hydrophilic and hydrophobic drugs. The degradation of MnO2 can be well controlled by adjusting the thickness of polydopamine film, and the strong π-π interactions between them also establish a strong fluorescence resonance energy transfer system, which allows us unprecedentedly to conveniently monitor drug loading process under naked eyes just by judging solution color changes. Additionally, in vitro experimental results demonstrate that the co-drug loaded system exhibits outstanding cancer therapy effect than single therapy, and we also report its first experimental example for effectively anti-cancer migration. The novel biomimetic synthesis method, effective co-loading different drugs with a visual pattern, and much better anticancer and anti-migration effect make our work potentially valuable in many fields.

One-step G-quadruplex-based fluorescence resonance energy transfer sensing method for ratiometric detection of uracil-DNA glycosylase activity

Uracil-DNA glycosylase (UDG) is a crucial enzyme in base excision repair (BER) pathway. It can repair the uracil-induced DNA lesions and maintain the integrity of genome. In this paper, we developed a facile and ratiometric strategy for UDG activity detection using fluorescence resonance energy transfer (FRET). One double-stranded DNA (dsDNA) substrate consisting of strand 1 (dual-fluorescent dye-modified G-quadruplex sequence single-stranded DNA (ssDNA)), carboxyfluorescein (FAM) acted as donor and tetramethylrhodamine (TAMRA) as acceptor) and strand 2 (the complementary sequence of strand 1 containing three mismatched bases and three uracil bases) was introduced. When the UDG-catalyzed uracil is removed from dsDNA, the thermo-stability of dsDNA is decreased and the dual-fluorescent dye-modified G-quadruplex sequence ssDNA is released. Then, the ssDNA transforms into a G-quadruplex comformation, which brings the labeled FAM and TAMRA into close proximity, resulting in a strong FRET signal. In the absence of UDG, the relatively stable dsDNA separates the labeled FAM and TAMRA, giving a weak FRET signal. Thus, by measuring the system fluorescence intensity and exploiting FRET signal difference, UDG activity can be detected in a simple process. The detection limit is 0.087 U/mL without requiring additional signal amplification process. Besides, our developed strategy can also be used for screening the UDG inhibitors in a ratiometric fluorescence detection way.

A DNA tetrahedron nanoprobe-based fluorescence resonance energy transfer sensing platform for intracellular tumor-related miRNA detection.

Accurate and sensitive detection of disease-related microRNAs (miRNAs) is of great significance for early disease diagnosis. In this work, a DNA tetrahedron nanoprobe (DTNP)-based fluorescence resonance energy transfer (FRET) sensing platform (termed DTNP sensor) was constructed for sensitive detection of tumor-related miRNA (e.g., hsa-miR-146b-5p) with DNA assisted cyclic amplification. DTNP was synthesized by DNA self-assembly. In the absence of hsa-miR-146b-5p, the fluorescence DNA (HP) modified with FAM at the 5' terminal and TAMRA at the 3' terminal cannot form the hairpin structure because of the hybridization with the extended DNA strand of the DNA tetrahedron, resulting in a low FRET effect. In the presence of hsa-miR-146b-5p, it would complementarily hybridize with the extended DNA strand of the DNA tetrahedron, leading to the release of HP and occurrence of strong FRET. Thus, the concentration of hsa-miR-146b-5p can be revealed by the change in the fluorescence intensity. Moreover, an assistant DNA was employed to replace hsa-miR-146b-5p for cyclic signal amplification, which can further enhance the detection sensitivity. Under the optimal experimental conditions, the limit of detection for hsa-miR-146b-5p was as low as 6 pM (S/N = 3). Furthermore, the DTNP sensor was successfully applied to evaluate the hsa-miR-146b-5p expression levels in different cell lines. The inhibition of hsa-miR-146b-5p expression in different cells was also investigated and a satisfactory result was obtained.

A novel and sensitive ratiometric fluorescence assay for carbendazim based on N-doped carbon quantum dots and gold nanocluster nanohybrid

A novel fluorescence “turn on” ratiometric fluorescent sensor was employed to determine carbendazim. The sensing process was achieved through the strong fluorescence resonance energy transfer (FRET) between nitrogen doped carbon quantum dots (N-CQDs) and gold nanocluster (AuNCs). The photoluminescence intensity of N-CQDs can be deactivated by AuNCs through FRET effect and recovered by the addition of carbendazim. The ratiometric detection of carbendazim is achieved by recording the photoluminescence and second-order Rayleigh scattering (SRS) signal of N-CQDs/AuNCs system. With the introduction of carbendazim to the sensing platform resulted in the photoluminescence and SRS signal of N-CQDS/AuNCs enhancing. UV–vis absorption, Zeta potential and fluorescence lifetime analyses indicate that the fluorescence turn on process can be attributed to the aggregation of AuNCs breaks the FRET process and increases SRS intensity. N-CQDs/AuNCs probe present a good sensitivity and selectivity for carbendazim detection, with two linear response ranges (1−100 μM, 150−1000 μM), low detection limit of 0.83 μM and 37.25 μM. Furthermore, real sample analyses indicate that the as-presented sensor has potentials in carbendazim determination in real sample analyses.

A smart bioresponsive nanosystem with dual-modal imaging for drug visual loading and targeted delivery

Abstract Most of present studies are focusing on improving cancer therapy effect by synthesizing new carriers or drugs, while little attention is paid to develop new drug loading patterns and improve the efficiency. Herein, a novel and smart nanosystem that not only realized targeted delivery and responsive degradation but also achieved facilely visual drug loading and release, was reported for the first time. To build this system, highly fluorescent carbon quantum dots (CQDs) were synthesized and served as an effective indicator and carrier to achieve visual doxorubicin (DOX) loading by the strong fluorescence resonance energy transfer (FRET) interactions between them. Biodegradable nano-MnO2 was then synthesized by biomineralization with folic acid modified bovine serum albumin, which was employed as the other fluorescence quencher for CQDs and DOX and established the other FRET system to visually monitor drug loading. The double-FRET nanosystem could be specifically ingested by cancer cells and responsively degraded by intracellular H+/H2O2 or glutathione. Meanwhile, this triggered drug release behavior was visually detected after gradual dissociation of the two FRET systems by monitoring the fluorescence and magnetic resonance changes. More importantly, both in vitro and in vivo experiments demonstrated that the novel nanosystem exhibited much better cancer therapy effects than pure DOX, and we also reported its first experimental example to treat cancer migration. This work established a new pattern to improve and monitor drug loading, delivery and release, and demonstrated its good performance in cancer therapy and anti-migration.

Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay.

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

FRET-based analysis and molecular modeling of the human GPN-loop GTPases 1 and 3 heterodimer unveils a dominant-negative protein complex.

GPN-loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1-Gpn3 interaction in live HEK293-AD cells. Transiently expressed EYFP-Gpn1 and Gpn3-CFP generated a strong FRET signal, indicative of a very close proximity, in the cytoplasm of HEK293-AD cells. Molecular modeling of the human Gpn1-Gpn3 heterodimer based on the crystallographic structure of Npa3, the Saccharomyces cerevisiae Gpn1 ortholog, revealed that human Gpn1 and Gpn3 associate through a large interaction surface formed by internal α-helix 7, insertion 2, and the GPN-loop from each protein. In site-directed mutagenesis experiments of interface residues, we identified the W132D and M227D EYFP-Gpn1 mutants as defective to produce a FRET signal when coexpressed with Gpn3-CFP. Simultaneous but not individual expression of Gpn1 and Gpn3, with either or both proteins fused to EYFP, retained RNAPII in the cytoplasm and markedly inhibited global transcription in HEK293-AD cells. Interestingly, the W132D and M227D Gpn1 mutants that showed an impaired ability to interact with Gpn3 by FRET were also unable to delocalize RNAPII in this assay, indicating that an intact Gpn1-Gpn3 interaction is required to display the dominant-negative effect on endogenous Gpn1/Gpn3 function we described here. Altogether, our results suggest that a Gpn1-Gpn3 strong interaction is critical for their cellular function.

Drug screening assay based on the interaction of intact Keap1 and Nrf2 proteins in cancer cells.

The Nrf2-Keap1 interaction is the major regulatory pathway for cytoprotective responses against oxidative and electrophilic stresses. Keap1, a substrate protein of a Cul3-dependent E3 ubiquitin ligase complex, is a negative regulator of Nrf2. The use of chemicals to regulate the interaction between Keap1 and Nrf2 has been proposed as a strategy for the chemoprevention of degenerative diseases and cancers. The interactions between Keap1 and Nrf2 in vitro and in vivo were investigated using fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) strategies in our study. Nrf2 with its N-terminal fused to eGFP and Keap1 with its C-terminal fused to mCherry were expressed and purified in vitro. When purified eGFP-Nrf2 and Keap1-mChrry proteins were mixed together, a strong FRET signal could be detected, indicating an efficient energy transfer from eGFP to mCherry. Moreover, the FRET was detected in vivo using confocal microscopy in colon cancer HCT-116 cells that were co-transfected with eGFP-Nrf2 and Keap1-mCherry. Finally, using an eGFP BiFC approach, the Keap1-Nrf2 interaction was also detected in MCF7 cells by transfecting eGFP N-terminal fused to Nrf2 (eN158-Nrf2) and eGFP C-terminal fused to Keap1 (eC159-Keap1). Using the BiFC and FRET systems, we demonstrated that the prototypical Nrf2-activiting compound tBHQ and the antitumor drug F-dUrd might interfere with the intracellular interaction between Keap1 and Nrf2 whereas the 5-Fu have little role in activating the protective response of Nrf2 pathway in cancer cells. By analyzing the perturbation of the energy transfer between the donor and acceptor fluorophores and the bimolecular fluorescence complementation of eGFP, we can screen potential inhibitors for the interaction between Keap1 and Nrf2.

Label-free detection of histone based on cationic conjugated polymer-mediated fluorescence resonance energy transfer

A simple and homogeneous histone assay is developed based on histone-induced DNA compressing coupled with cationic conjugated polymer (CCP)-mediated fluorescence resonance energy transfer (FRET). In this strategy, the CCP serves as the FRET donor and SYBR Green I (SG), which can strongly fluoresce not at its free state but after intercalated into the double stranded calf thymus DNA (dsDNA), serves as the acceptor of FRET. In the absence of histone, the dsDNA-SG and CCP combine with each other through electrostatic interaction and the strong FRET from CCP to SG occurs due to the overlapping between the fluorescent emitting spectrum of the CCP and the absorption spectrum of SG. Upon the introduction of histone, the formed compact complex of histone/dsDNA will lead to the compression of dsDNA structure and prevent SG binding to dsDNA and fluorescing, which gives rise to a significant decrease of FRET efficiency between CCP and SG. Thus, the quantitative analysis of histone is realized by monitoring the change of FRET ratio, namely, the intensity ratio of the two emission bands of CCP and SG. Due to the light harvesting and fluorescence amplification properties of CCP, high sensitivity is achieved with a low detection limit of 0.74ng/mL histone. This strategy provides a simple, homogeneous and sensitive strategy for histone analysis in the study of histone-related biological processes.

Universal fluorometric aptasensor platform based on water-soluble conjugated polymers/graphene oxide

We designed a universal and sensitive fluorometric aptasensor that uses a fluorescence resonance energy transfer (FRET) mechanism. In the aptasensor, water-soluble conjugated poly(9,9-bis(6'-N,N,N-trimethylammonium)hexyl)fluorine phenylene (PFP) is used as the energy donor and a carboxyfluorescein (FAM)-labeled aptamer is used as the energy acceptor. Graphene oxide (GO) used as a quencher can specifically adsorb the aptamer, leading to quenching of the FAM fluorescence. In the presence of targets, the aptamer can change its conformation to prevent adsorption by GO. Strong FRET was thus obtained owing to the electrostatic interactions between PFP and the aptamer. In contrast, in the absence of targets, the FRET was weak because of GO specifically adsorbing the aptamer and quenching the fluorescence. Bisphenol A (a pollutant molecule) and dopamine (a biomolecule) were used as models to successfully validate the feasibility, universality, and high selectivity and sensitivity of this aptasensor. This method can detect BPA at environmentally relevant concentrations (less than 1 ng/mL) with a limit of detection of 0.005 ng/mL. A low limit of detection (1.0 nmol/L) was also obtained for dopamine. In addition, this aptasensor is applicable in real samples and in diluted human plasma and human serum. Good recovery rates from 95% to 105% and from 95% to 107% were obtained for bisphenol A and dopamine, respectively. Furthermore, adenosine detection was successfully achieved by the same mechanism, proving the universality. It is expected that the aptasensor could be applied in detecting other contaminants, biomolecules, and heavy metal ions by a change in only the aptamer sequence. Graphical abstract A universal and sensitive fluorometric aptasensor was developed for the detection of small molecules that uses a fluorescence resonance energy transfer (FRET) mechanism. GO graphene oxide. PFP poly(9,9-bis(6'-N,N,N-trimethylammonium)hexyl)fluorine phenylene.

FRET study in oligopeptide-linked donor–acceptor system in PVA matrix

An oligopeptide: Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys-NH2, cleaved specifically by a matrix metalloproteinase 9 (MMP-9) at the Ser-Leu bond, was labeled on the ε-NH2 groups of lysine with donor (5, 6 TAMRA) and acceptor (HiLyte647) dye. The donor control was a peptide labeled with 5, 6 TAMRA only on the C-terminal lysine, and the acceptor control was free HiLyte647. Following three products were studied by dissolving in 10% (w/w) poly(vinyl alcohol) and dried on glass slides forming 200 micron films. Absorption spectra of the films show full additivity of donor and acceptor absorptions. A strong Fluorescence Resonance Energy Transfer (FRET) with an efficiency of about 85% was observed in the fluorescence emission and excitation spectra. The lifetime of the donor was shorter and heterogeneous compared with the donor control.

Interactions of amelogenin with phospholipids.

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (∼334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.

Adenosine deaminase biosensor combining cationic conjugated polymer-based FRET with deoxyguanosine-based photoinduced electron transfer.

We demonstrated a sensitive and selective adenosine deaminase (ADA) detection by modulating the fluorescence resonance energy transfer (FRET) between cationic conjugated poly(9,9-bis(6'-N,N,N-trimethylammonium) hexyl)fluorine phenylene) (PFP) and the deoxyguanosine-tailored hairpin aptamer. The hairpin aptamer was labeled with a fluorophore FAM at one end and three deoxyguanosines (Gs) at the other end as a quencher. In the absence of ADA, aptamer forms hairpin-like conformation with adenosines making close affinity of Gs and FAM, which results in the weak FRET from PFP to FAM because of FAM fluorescence being quenched by Gs via photoinduced electron transfer (PET). After addition of ADA, adenosine was hydrolyzed by ADA, followed by the release of free aptamer. In this case, FAM being far away from Gs, the strong FRET thus was obtained due to the quenching process being blocked. Therefore, the new strategy based on the FRET ratio enhancement is reasonably used to detect the ADA sensitively, combining the fluorescence signal amplification of conjugated polymers with the initiative signal decreasing by Gs. The detection limit of the ADA assay is 0.3 U/L in both buffer solution and human serum, which is more sensitive than most of those previously documented methods. Importantly, the assay is rapid, homogeneous, and simple without a complicated treating process. The ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA), was also studied based on this assay, and the detection limit of EHNA is 10 pM. This strategy provides a new platform for the detection of other biomolecules and enzymes.

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

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).

In Situ Measurement of RyR2-Calmodulin Binding in Permeabilized Cardiomyocytes

Calmodulin (CaM) is a Ca-sensing protein and important regulator to RyR2. Defective CaM association with and regulation of RyR2 has been implicated in cardiac hypertrophy, heart failure and Catecholaminergic Polymorphic Ventricular Tachycardia. However, direct measurement of the RyR-CaM binding properties in myocytes is lacking despite its potential clinical importance. This is most likely due to the interference from CaM's multiple binding targets, such as L-type Ca channel, Calcineurin, and CaMKII. Fluorescence Resonance Energy Transfer (FRET) was used to measure the RyR2-specific bound CaM in permeabilized rat and mouse cardiomycytes. FKBP12.6 and CaM bind to RyR2 in close physical proximity so that strong FRET can be detected between fluorescently labeled FKBP12.6 (F-FKBP12.6) and F-CaM. To confirm the specificity of CaM binding to RyR2 and measure the percentage of CaM visible at the Z-line is RyR-bound under physiological conditions, we used mutant RyR2 knock-in mice expressing a triple mutation in the RyR2 (RyR2ADA). The mutation prevents CaM binding to RyR2 but should not alter binding of CaM to other targets. Our data showed that 1) the apparent Kd of CaM to RyR in rat cardiac myocytes (at 100 nM [Ca]) was 18.2± 2nM (n=XX); 2) heterozygous RyR2ADA/+ mutation decreased the maximal binding sites (on RyR2) by 50% without appreciably altering affinity in mouse myocytes (Kd =16.9 ±XX nM in RyR2ADA/+ and 13.4 ± XX nM, in WT mice, p< 0.05); 3) The total CaM binding visible at the Z-line was reduced by 45%, implying that ∼90% of Z-line CaM is RyR2 bound. These results are the first quantitative in situ RyR2-CaM binding measurements.

AKAP79/150 Signal Complexes in G-Protein Modulation of Neuronal Ion Channels

Voltage-gated M-type (KCNQ) K+ channels play a critical role in modulation of neuronal excitability and action potential firing. A-kinase-anchoring protein (AKAP)79/150 mediated PKC phosphorylation of M channels is involved in M current (IM) suppression by muscarinic M1, but not bradykinin B2 receptors. In this study, we first explored if purinergic and angiotensin suppression of IM in superior cervical ganglion (SCG) sympathetic neurons involves AKAP79/150. Transfection into rat SCG neurons of ΔA-AKAP79, which lacks the A-domain necessary for PKC binding, or the absence of AKAP150 in AKAP150-/- mice, did not affect IM suppression by the purinergic agonist UTP, nor by bradykinin, but did reduce IM suppression by muscarinic agonists and by angiotensin II. Transfection of AKAP79, but not ΔA-AKAP79 or AKAP15, “rescued” the muscarinic suppression of IM in AKAP150-/- neurons. We also tested association of AKAP79 or KCNQ channels with M1, B2, P2Y6 and AT1 receptors via fluorescence resonance energy transfer (FRET) experiments on CHO cells under total internal refection fluorescence microscopy, which revealed substantial FRET between AKAP79 with M1 and AT1 receptors, but only weak FRET with P2Y6 or B2 receptors. Similarly, we observed strong FRET between KCNQ2 with M1 and AT1, but not P2Y6 or B2, receptors. The involvement of AKAP79/150 in the regulation of N- and L-type Ca2+ channels in SCG neurons by Gq/11-coupled muscarinic receptors and by cAMP/PKA was also studied. We found AKAP79/150 to not play a role in the former, but to be necessary for forskolin-induced up-regulation of the L-type current. Our data suggest that AKAP79/150 orchestrates signal complexes that include PKC, KCNQ subunits and M1 or AT1 receptors, which correlates with the PIP2-depletion mode of neuronal M current suppression, but does not generalize to Gq/11-mediated inhibition of N- or L-type Ca2+ channels.

Bidirectional Fluorescence Resonance Energy Transfer (FRET) in Mutated and Chemically Modified Yellow Fluorescent Protein (YFP)

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Förster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.