Donor Probes Research Articles

Ratiometric electrochemiluminescence sensing and intracellular imaging of ClO- via resonance energy transfer.

Electrochemiluminescence resonance energy transfer (ECL-RET) is a versatile signal transduction strategy widely used in the fabrication of chem/biosensors. However, this technique has not yet been applied in visualized imaging analysis of intracellular species due to the insulating nature of thecell membrane. Here, we construct a ratiometric ECL-RET analytical method for hypochlorite ions (ClO-) by ECL luminophore,with a luminol derivative (L-012) as the donor and afluorescence probe (fluorescein hydrazide) as the acceptor. L-012 can emit astrong blue ECL signal and fluorescein hydrazide has negligible absorbance and fluorescence signal in the absence of ClO-. Thus, theECL-RET process is turned off at this time. In the presence of ClO-, however, the closed-loop hydrazide structure in fluorescein hydrazide is opened via specific recognition with ClO-, accompanied with intensified absorbance and fluorescence signal. Thanks to the spectral overlap between theECL spectrum of L-012 and theabsorption spectrum of fluorescein, theECL-RET effect isgradually recovered with the addition of ClO-. Furthermore, the ECL-RET system has been successfully applied to image intracellular ClO-. Although the insulating nature of thecell itself can generate ashadow ECL pattern in the cellular region, extracellular ECL emission penetrates thecell membrane and excites intracellular fluorescein generated by the reactions between fluorescein hydrazide and ClO-. The cell imaging strategy via ECL-RET circumvents the blocking of thecell membrane and enables assays of intracellular species. The importance of theECL-RET platform lies in calibrating the fluctuation from theexternal environment and improving the selectivity by using fluorescent probes. Therefore, this ratiometric ECL sensor has shown broad application prospects in the identification of targets in clinical diagnosis and environmental monitoring.

Immunoassay System Based on the Technology of Time-Resolved Fluorescence Resonance Energy Transfer.

To enhance the specificity and sensitivity, cut the cost, and realize joint detection of multiple indicators, an immunoassay system based on the technology of time-resolved fluorescence resonance energy transfer (TR-FRET) was studied. Due to the FRET of the reagent, the donor probe and acceptor probe emitted specific fluorescence to enhance specificity. Long-lifetime specific fluorescence from the acceptor probe was combined with time-resolved technology to enhance sensitivity. A xenon flash lamp and a photomultiplier tube (PMT) were selected as the light source and detector, respectively. A filter-switching mechanism was placed in the light path, so the fluorescence signal from the donor and acceptor was measured alternately. The instrument's design is given, and some specificI parts are described in detail. Key technical specifications of the instrument and procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6(IL-6) were tested, and the test results were presented subsequently. The CV value of the self-designed counting module is better than 0.01%, and the instrument noises for 620 nm and 665 nm are 41.44 and 10.59, respectively. When set at 37 °C, the temperature bias (B) is 0.06 °C, and the temperature fluctuation is 0.10 °C. The CV and bias are between ±3% and 5%, respectively, when pipetting volumes are between 10 μL and 100 μL. Within the concentration range of 0.01 nM to 10 nM, the luminescence values exhibit linear regression correlation coefficients greater than 0.999. For PCT detection, when the concentration ranges from 0.02 ng/mL to 50 ng/mL, the correlation coefficient of linear fitting exceeds 0.999, and the limit of quantification is 0.096 ng/mL. For CRP and IL-6, the detection concentration ranges from 0 ng/mL to 500 ng/mL and 0 ng/mL to 20 ng/mL, respectively, with limits of quantification of 2.70 ng/mL and 2.82 ng/mL, respectively. The experimental results confirm the feasibility of the technical and instrumental solutions.

ONOO- Activatable Fluorescent Sulfur Dioxide Donor for a More Accurate Assessment of Cell Ferroptosis.

Ferroptosis is critical in the treatment of tumor therapies. Thus, monitoring reactive oxygen species (ROS) is of great significance for accurate assessment in ferroptosis without any interference. However, current probes for monitoring ROS during ferroptosis suffer from a drawback in that the probes consume ROS during detection, which inhibits the ferroptosis process and thus affects the accuracy and effectiveness of monitoring the process of ferroptosis. Herein, a new fluorescent donor probe, TFMU-SO2D, with the combination of the moiety of the SO2 donor is designed and synthesized by introducing the aryl boronate moieties that could give it the ability to effectively recognize ONOO-. The released SO2 could consume excess glutathione and regulate oxidative stress by elevating ROS levels, which would offset the ROS depletion by TFMU-SO2D and ensure accuracy in monitoring the ferroptosis process. The experimental results demonstrated that TFMU-SO2D possessed satisfactory performance for monitoring ONOO- as well as simultaneously releasing SO2 in oxidative stress stimulated by monensin and ferroptosis stimulated by erastin and RSL3. Additionally, the capability of SO2 synergized with ferroptosis to inhibit the viability of cancer cells was demonstrated by the CCK8 assay, which may be due to the fact that SO2 can potentiate ferroptosis cell death by increasing the ROS level. Overall, these combined results indicated that TFMU-SO2D possesses the excellent ability to precisely monitor ONOO- during ferroptosis without interference, which is significant for accurately accessing ferroptosis, cancer treatment, and drug development.

Defining the structure and orientation of the N-terminal domain of cardiac troponin T in relationship to actin.

Cardiac troponin T (cTnT) serves as the tropomyosin (Tm) binding subunit of the troponin complex and, when mutated, it often leads to pathologic cardiac remodeling observed in patients with hypertrophic and dilated cardiomyopathies. Part of the N-terminus of cTnT is responsible for positioning Tm along the actin groove and it immediately flanks the Tm overlap domain. Understanding the molecular mechanisms underlying these observations has been hindered by the absence of a high-resolution structure in the highly flexible N-terminal domain. A recently published cryo-electron-microscopy structure shows interactions between the N-terminus of cTnT, around amino acid 87, and the F-actin backbone; however, this interaction was not observed in calcium saturated conditions. Here, we employ single-donor dual-acceptor time-resolved fluorescence resonance energy transfer (TR-FRET) to probe the positioning of the cTnT N-terminal extension in fully reconstituted cardiac thin filaments (CTF). We hypothesize the N-terminal tail interacts with F-actin in the −Ca2+ condition aiding in stabilizing Tm in the blocked position. Conversely, we hypothesize that the that the N-terminal tail is “fly-casting” and not interacting with F-actin in +Ca2+ condition. To test this, a donor probe (IAEDANS) was placed on either the Cys-substituted residues N100C, N73C and M60C of cTnT and an acceptor probe (DABMI) was placed on the endogenous Cys-374 of actin. Preliminary data revealed two measurable discrete distances for all residues, one short and one long, indicating that FRET occurred with two actin monomers. Initial results suggest that the N-terminal tail is interacting with F-actin in high and low calcium conditions. Continuing work aims to probe additional sites on the N-terminus, specifically A20C and A2C. Resolving the structure and orientation of the N-terminal domain of cTnT is critical to understanding the regulatory mechanisms of the CTF.

Turn-on detection of Al3+ and Zn2+ ions by a NSN donor probe: reversibility, logic gates and DFT calculations.

An efficient dual functional naphthalene-derived Schiff base NpSb probe has been synthesised and evaluated for its fluorescence and chromogenic response towards metal ions. The NpSb probe was capable of selectively recognising Al3+ and Zn2+ ions when they were excited at the same wavelength in an aqueous organic solvent system. Almost non-fluorescent NpSb displayed a 'turn-on' fluorescence response when treated with Zn2+ (λem = 416 nm) and Al3+ (λem = 469 nm) ions due to the chelation-enhanced fluorescence (CHEF) effect. The limit of detection (LoD) values for Al3+ and Zn2+ have been determined to be 38.0 nM and 43.0 nM, respectively. The binding constants for Al3+ and Zn2+ were found to be 1.18 × 106 M-1 and 3.5 × 105 M-1, respectively. The NpSb also acted as a colorimetric sensor for Al3+ as the colour of the probe's solution turned to pale green from colourless upon Al3+ addition. The binding mechanism between NpSb and Zn2+/Al3+ was supported by the ESI-MS, Job's plot, NMR, and DFT studies. The reversibility experiments were carried out with an F- ion and EDTA with the development of corresponding logic gates. Moreover, NpSb could be applied to detect Al3+ ions in real samples such as tap water, distilled water and soil samples.

Glutathione-Capped CdTe Quantum Dots Based Sensors for Detection of H2O2 and Enrofloxacin in Foods Samples.

Additives and antibiotic abuse during food production and processing are among the key factors affecting food safety. The efficient and rapid detection of hazardous substances in food is of crucial relevance to ensure food safety. In this study, a water-soluble quantum dot with glutathione as a ligand was synthesized as a fluorescent probe by hydrothermal method to achieve the detection and analysis of H2O2. The detection limits were 0.61 μM in water and 68 μM in milk. Meanwhile, it was used as a fluorescent donor probe and manganese dioxide nanosheets were used as a fluorescent acceptor probe in combination with an immunoassay platform to achieve the rapid detection and analysis of enrofloxacin (ENR) in a variety of foods with detection limits of 0.05-0.25 ng/mL in foods. The proposed systems provided new ideas for the construction of fluorescence sensors with high sensitivity.

Conformational Rearrangements in the Redox Cycling of NADPH-Cytochrome P450 Reductase from Sorghum bicolor Explored with FRET and Pressure-Perturbation Spectroscopy.

Simple SummaryNADPH-cytochrome P450 reductase (CPR) enzymes are known to undergo an ample conformational transition between the closed and open states in the process of their redox cycling. To explore the conformational landscape of CPR from the potential biofuel crop Sorghum bicolor (SbCPR), we incorporated a FRET donor/acceptor pair into the enzyme and employed rapid scanning stop-flow and pressure perturbation spectroscopy to characterize the equilibrium between its open and closed states at different stages of the redox cycle. Our results suggest the presence of several open conformational sub-states differing in the system volume change associated with the opening transition (ΔV0). Although the closed conformation always predominates in the conformational landscape, the population of the open conformations increases by order of magnitude upon the two-electron reduction and the formation of the disemiquinone state of the enzyme. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV0) of the opening transition. While the ΔV0 characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.

Design of a ratiometric fluorescence sensor based on metal organic frameworks and Ru(bpy)32+-doped silica composites for 17β-Estradiol detection

17β-Estradiol (E2), an important endocrine disrupting compound, could be quantitatively detected by fluorescence resonance energy transfer (FRET) aptasensor, designed in this paper. Metal organic frameworks have large specific surface area and easily modifiable groups, which are helpful for the construction of aptasensor. Specifically, streptavidin was immobilized on the synthesized MIL-53-NH2 by covalent bonding, and further linked with the biotin modified E2 aptamer (apt) through specific bonding between avidin and biotin to obtain the FRET donor probe (MIL-53-apt). Meanwhile, complementary DNA (cDNA) modified Ru(bpy)32+-doped silica nanoparticles (RuSiO2-cDNA) were prepared through covalent bonding. They acted as the FRET acceptor probe, since its absorption spectrum showed large overlap with the emission spectrum of MIL-53-apt. In the presence of E2, aptamer modified donor probes tended to bind with E2, owing to their higher selectivity and affinity. Therefore, the optimal distance between FRET pairs was broken, resulting in the fluorescence emission recovery of donor and the fluorescence emission of acceptor decreased. Under optimal conditions, this proposed aptasensor displayed sensitive detection of E2 ranging from 0.5 to 1000 nM with a detection limit of 0.2 nM. Furthermore, the sensor provides a promising method for rapid and sensitive detection of other small biological molecules.

Fluorescent ZnO Quantum Dot Probe to Study Glucose–Glucose Oxidase Interaction via Fluorescence Resonance Energy Transfer

In this work, emphasis is to utilize FRET method to analyze GOx and glucose interaction using fluorescent ZnO QDs as the probes. Fluorescent ZnO and ZnOext QDs were synthesized using sol– gel technique and green synthesis method as donor and acceptor nanoprobes. Structural, optical and morphological characterization of QDS were carried out using UV-visible absorption, fluorescence, FTIR, XRD and field emission scanning electron microscopy. Band gap estimated from Tauc plot is 3.49 and 3.35 eV for ZnO and ZnOext QDs and XRD reveal Wurtzite structure of grown crystals. Systematic absorption study for ZnO–GOx and ZnOext–GOx reveal association constant of –8.3361 M–1 and –2.57646 M –1 for ZnO and ZnOext using Benesi-Hildebrand plot. The binding constant obtained from Stern-Volmer equation is 1.0466 μM –1 and 1.97 μM –1 for ZnO/ZnOext–GOx conjugate and suggest static quenching in the system. CD spectroscopy reveal native state of protein in conjugate sytem. Constant Förster radius (Ro) in ZnOext–GOx system suggest the average 〈κ2〉 as 2/3 and is independent of donor–acceptor distance as normally assumed in FRET system varies from ∼4.5% in ZnO–GOx and ∼5% in ZnOext–GOx system. It indicate better energy transferrin earlier system than formal. The physiological range of glucose from 60 mg–440 mg/dl using this technique in which ZnOext–GOx as donor probe exhibit better response towards glucose than ZnO–GOx system and correlated to the presence of phyotochemicals on the QD surface.

Pulmonary surfactant protein SP-B nanorings induce the multilamellar organization of surfactant complexes

Surfactant protein SP-B is absolutely required for the generation of functional pulmonary surfactant, a unique network of multilayered membranes, which stabilizes the respiratory air-liquid interface. It has been proposed that SP-B assembles into hydrophobic rings and tubes that facilitate the rapid transfer of phospholipids from membrane stores into the interface and the formation of multilayered films, ensuring the stability of the alveoli against physical forces leading to their collapse. To elucidate the molecular organization of SP-B-promoted multilamellar membrane structures, time-resolved Förster Resonance Energy Transfer (FRET) experiments between BODIPY-PC or BODIPY-derivatized SP-B (BODIPY/SP-B), as donor probes, and octadecylrhodamine B, as acceptor probe, were performed in liposomes containing SP-B or BODIPY/SP-B. Our results show that both SP-B and fluorescently labeled SP-B oligomers mediate the connection of adjacent bilayers. Furthermore, by applying rational models to the FRET data, we have been able to provide quantitative details of the structure of SP-B-induced multilayered membrane arrays at the nanometer scale, defining interactions between SP-B rings as key elements for connecting surfactant membranes. The data sustain the structural model and the mechanism of action of SP-B assemblies to sustain the crucial surfactant function.

Two fluorescence quenching immunochromatographic assays based on carbon dots and quantum dots as donor probes for the determination of enrofloxacin

Two fluorescence quenching lateral flow immunochromatographic assays were developed based on carbon dots (CDs) and quantum dots (QDs) as donor signal probes.

Clopidogrel as a donor probe and thioenol derivatives as flexible promoieties for enabling H2S biomedicine

Hydrogen sulfide has emerged as a critical endogenous signaling transmitter and a potentially versatile therapeutic agent. The key challenges in this field include the lack of approved hydrogen sulfide-releasing probes for in human exploration and the lack of controllable hydrogen sulfide promoieties that can be flexibly installed for therapeutics development. Here we report the identification of the widely used antithrombotic drug clopidogrel as a clinical hydrogen sulfide donor. Clopidogrel is metabolized in patients to form a circulating metabolite that contains a thioenol substructure, which is found to undergo spontaneous degradation to release hydrogen sulfide. Model studies demonstrate that thioenol derivatives are a class of controllable promoieties that can be conveniently installed on a minimal structure of ketone with an α-hydrogen. These results can provide chemical tools for advancing hydrogen sulfide biomedical research as well as developing hydrogen sulfide-releasing drugs.

Energy-Transfer Metal-Organic Nanoprobe for Ratiometric Sensing with Dual Response to Peroxynitrite and Hypochlorite.

An energy-transfer metal–organic nanoprobe is designed for ratiometric sensing with dual response to both peroxynitrite (ONOO–) and hypochlorite (ClO–). Here, a nanoscale metal–organic framework (NMOF) acts as the energy donor and molecular probe as the acceptor to construct a Förster resonance energy transfer (FRET) nanosystem. Biocompatible dextran conveniently binds to the NMOF surface through multiple weak coordination interactions to improve water dispersibility and cell uptake. Dextran can also coordinate with the molecular probe with arylboronic acid group, which enables the convenient grafting of molecular probes to the NMOF surface to construct energy-transfer nanoprobes. Because of efficient FRET, the bright blue fluorescence of NMOF is quenched, whereas red emission from the acceptor is enhanced. Upon reacting with ONOO–, the probe departs from NMOF and the fluorescence of NMOF is recovered because of the interruption of FRET. When reacting with ClO–, the phenothiazine moiety in the molecular probe is oxidized into phenothiazine-5-oxide, which leads to more efficient energy transfer and the fluorescence shifts from red to orange. The nanoprobes are also successfully applied to the detection of ONOO– and ClO– in living cells.

Influence of stabilizing components on the integrity of antitumor liposomes loaded with lipophilic prodrug in the bilayer.

Previously, we proposed a liposomal formulation of melphalan (Mlph)-a chemotherapeutic alkylating agent-incorporated in a fluid lipid bilayer in the form of dioleoylglyceride ester. In this work, we compared the stabilizing effect of different amphiphiles included in the Mlph-liposomes, such as phosphatidylinositol (PI), ganglioside GM1, a conjugate of N-carboxymethyl-modified oligoglycine with dioleoylphosphatidylethanolamine (acidic lipopeptide), and polyethylene glycol (2000 Da) conjugated with dipalmitoylphosphatidylethanolamine (PEG-lipid), upon incubation in human serum. Mean hydrodynamic diameter values (86-90 nm) were similar among different liposome samples, while zeta potential values considerably varied. The formulations were incubated in human serum at 37 °C for different time intervals up to 24 h. Liposome integrity was evaluated by changes in fluorescence upon leakage of calcein or disruption of Förster resonance energy transfer between donor and acceptor fluorescent lipid probes in the bilayer. The best stabilization of liposomes was achieved upon the addition of ganglioside GM1 or the acidic lipopeptide. Inclusion of 10 mol% PI improved liposome stability only for the first 4 h of incubation. Pegylated liposomal formulations of melphalan lipophilic prodrug with fluid phase bilayer were the least stable, which is probably due to the propensity of the PEG-lipid to exit liposome membranes. Cholesterol-containing bilayers of liquid ordered phase, supplemented with sufficient amounts of the PEG-lipid, showed good stability in serum.

Aptamer based fluorometric determination of ATP by exploiting the FRET between carbon dots and graphene oxide.

The authors describe a fluorometric aptamer based assay for adenosine triphosphate (ATP). It is based on the use of carbon dots (CDs) and graphene oxide (GO). The resultant CD-aptamer is adsorbed on the surface of GO via π-stacking and hydrophobic interaction, and the fluorescence of CD-aptamer is quenched via fluorescence resonance energy transfer (FRET) between CDs and GO. If ATP is present, it will bind to the aptamer and the CD-aptamer will be desorbed from GO. This will suppress FRET and the fluorescence of the CDs is restored. Under the optimal conditions and at typical excitation/emission wavelengths of 358/455nm, the assay has a 80 pM detection limit and a linear range that extends from 0.10 to 5.0nM concentrations of ATP. The method was successfully applied to the determination of ATP in yogurt samples. This method can also be conceivably applied to the detection of other analytes for which appropriate aptamers are available. Graphical abstract Schematic of a novel fluorometric ATP assay based on the fluorescence resonance energy transfer (FRET) between aptamer modified carbon dots (CD-aptamer) and graphene oxide (GO). CD-aptamer was used as the energy donor and molecular recognition probe, and GO acted as energy acceptor. This assay exhibits high sensitivity and selectivity with a detection limit as low as 80 pM.

ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells

FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways. However, the signaling properties of fusion proteins are not consistent across GPCRs. In this study, we describe and characterize novel sensors based on the Systematic Protein Affinity Strength Modulation (SPASM) technique. Sensors consist of a GPCR and G protein tethered by an ER/K linker flanked by FRET probes. SPASM sensors are tested for the β2-, α1-, and α2- adrenergic receptors, and adenosine type 1 receptor (A1R), tethered to Gαs-XL, Gαi2, or Gαq subunits. Agonist stimulation of β2-AR and α2-AR increases FRET signal comparable to co-expressed FRET/BRET sensors. SPASM sensors also retain signaling through the endogenous G protein milieu. Importantly, ER/K linker length systematically tunes the GPCR-G protein interaction, with consequent modulation of second messenger signaling for cognate interactions. SPASM GPCR sensors serve the dual purpose of detecting agonist-induced changes in GPCR-G protein interactions, and linking these changes to downstream signaling.

Deciphering the Role of the Length of the Corona in Controlled NSET within Triblock Copolymers.

Nanometal surface energy transfer (NSET) from 2-anthracene sulfonate (2-AS) to gold nanoparticles in water-soluble triblock copolymers (TBP) P123 (PEO19PPO69PEO19) and F127 (PEO100PPO65PEO100) is systematically investigated. Fluorescence lifetime and anisotropy experiments provide thorough information on the locations of the 2-AS within these micelles. Variation in the size of the micellar corona region of the TBP is found to be the prime factor for different positions of 2-AS in them. Of late, nanometal surface energy transfer (NSET) from the donor probe to the surface of the metal nanoparticles has emerged as a potential tool for sensing and biolabeling. In the present work, the quenching of emission of the water-soluble 2-AS confined in the two different triblock copolymers in the proximity of a monolayer of the gold nanoparticles has been explored. Closer agreement between the experimental and theoretical characteristic distances has been found across the full wavelength range by the NSET approach. Understanding the location of the water-soluble dye in the vicinity of a polymeric drug delivery system is of significant importance, and how altered locations can trigger different controlled energy transfer efficiency from the 2-AS to the surfaces of gold nanoparticles (GNPs) has been discussed. This strategy could offer a new prospect in designing novel optical materials for chemical sensing and light harvesting endeavors.

Genetically encoded sensors of protein hydrodynamics and molecular proximity

The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1'-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼ 31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin.

Pharmacophore modeling using site-identification by ligand competitive saturation (SILCS) with multiple probe molecules.

Receptor-based pharmacophore modeling is an efficient computer-aided drug design technique that uses the structure of the target protein to identify novel leads. However, most methods consider protein flexibility and desolvation effects in a very approximate way, which may limit their use in practice. The Site-Identification by Ligand Competitive Saturation (SILCS) assisted pharmacophore modeling protocol (SILCS-Pharm) was introduced recently to address these issues, as SILCS naturally takes both protein flexibility and desolvation effects into account by using full molecular dynamics simulations to determine 3D maps of the functional group-affinity patterns on a target receptor. In the present work, the SILCS-Pharm protocol is extended to use a wider range of probe molecules including benzene, propane, methanol, formamide, acetaldehyde, methylammonium, acetate and water. This approach removes the previous ambiguity brought by using water as both the hydrogen-bond donor and acceptor probe molecule. The new SILCS-Pharm protocol is shown to yield improved screening results, as compared to the previous approach based on three target proteins. Further validation of the new protocol using five additional protein targets showed improved screening compared to those using common docking methods, further indicating improvements brought by the explicit inclusion of additional feature types associated with the wider collection of probe molecules in the SILCS simulations. The advantage of using complementary features and volume constraints, based on exclusion maps of the protein defined from the SILCS simulations, is presented. In addition, reranking using SILCS-based ligand grid free energies is shown to enhance the diversity of identified ligands for the majority of targets. These results suggest that the SILCS-Pharm protocol will be of utility in rational drug design.

Localization estimation of ionizing radiation-induced abasic sites in DNA in the solid state using fluorescence resonance energy transfer.

Clustered DNA damage is considered an important factor in determining the biological consequences of ionizing radiation. In this study, we successfully estimated the localization of abasic sites (APs) in DNA exposed to ionizing radiation using fluorescence resonance energy transfer (FRET) without any involvement of repair enzyme functions. A linearized plasmid (pUC19 digested by Sma I) was irradiated with: (60)Co γ rays; (4)He(2+) (2.0 MeV/u) particles; and the (12)C(5+) (0.37 MeV/u) particles in the solid state. A donor or acceptor fluorescent probe with a nucleophilic O-amino group was used to label APs. The results showed that the (12)C(5+) particle likely produced close APs within a track. The apparent distance calculated from the observed FRET efficiency (E) of around 0.10 was estimated to be approximately 17 base pairs. On the other hand, E values of (60)Co γ rays and the (4)He(2+) beam were less than those of the (12)C(5+) beam, increased with increasing AP density (the average number of APs per base pair), and were slightly greater than those of randomly distributed APs. We propose that the FRET method provides a degree of localization regardless of whether an AP cluster is single-stranded or bistranded DNA damage.