Fluorescence Energy Transfer Efficiency Research Articles

Fluorescence-Shadowing Nanoparticle Clusters for Real-Time Monitoring of Tumor Progression.

Monitoring tumor progression is important for elucidating appropriate therapeutic strategies in response to anticancer therapeutics. To fluorescently monitor the in vivo levels of tumor-specific enzymes, we prepared matrix metalloprotease (MMP)-responsive gold nanoparticle (AuNP) clusters to sense tumor microenvironments. Specifically, AuNPs and quantum dots (QDs) were surface-engineered with two poly(ethylene glycol) [PEG] shells and cyclooctyne moieties, respectively, for the copper-free click reaction. Upon "peeling off" of the secondary shell from the double-PEGylated AuNPs under MMP-rich conditions, shielded azide moieties of the AuNPs were displayed toward the QD, and those two particles were clicked into nanoparticle clusters. This consequently resulted in a dramatic size increase and fluorescence quenching of QDs via fluorescence energy transfer (FRET) due to the molecular proximity of the particles. We observed that FRET efficiency was modulated via changes in MMP levels and exposure time. Cancer cell numbers exhibited a strong correlation with FRET efficiency, and in vivo studies that employed solid tumor models accordingly showed that FRET efficiency was dependent on the tumor size. Thus, we envision that this platform can be tailored and optimized for tumor monitoring based on MMP levels in solid tumors.

Ultrafast DNA Sensors with DNA Framework-Bridged Hybridization Reactions.

Intracellular DNA-based hybridization reactions generally occur under tension rather than in free states, which are spatiotemporally controlled in physiological conditions. However, how nanomechanical forces affect DNA hybridization efficiencies in in-vitro DNA assays, for example, biosensors or biochips, remains largely elusive. Here, we design DNA framework-based nanomechanical handles that can control the stretching states of DNA molecules. Using a pair of tetrahedral DNA framework (TDF) nanostructured handles, we develop bridge DNA sensors that can capture target DNA with ultrafast speed and high efficiency. We find that the rigid TDF handles bind two ends of a single-stranded DNA (ssDNA) and hold it in a stretched state, with an apparent stretching length comparable to its counterpart of double-stranded DNA (dsDNA) via atomic force microscopy measurement. The DNA stretching effect of ssDNA is then monitored using single-molecule fluorescence energy transfer (FRET), resulting in decreased FRET efficiency in the stretched ssDNA. By controlling the stretching state of ssDNA, we obtained significantly improved hybridization kinetics (within 1 min) and hybridization efficiency (∼98%) under the target concentration of 500 nM. The bridge DNA sensors demonstrated high sensitivity (1 fM), high specificity (single mismatch mutation discrimination), and high selectivity (suitable for the detection in serum and blood) under the target concentration of 10 nM. Controlling the stretching state of ssDNA shows great potential in biosensors, bioimaging, and biochips applications.

A Versatile Photoinduced Electron Transfer‐Based Upconversion Fluorescent Biosensing Platform for the Detection of Disease Biomarkers and Nerve Agent

AbstractUpconversion nanoparticles (UCNPs) have emerged to be a new family of fluorescent probes for bioanalytical applications. In a typical design, the UCNPs act as the energy donors in a fluorescence resonance energy transfer (FRET) system, in which the target molecules mediate the energy transfer from the UCNPs to the acceptors, and their quantity information is consequently converted into the “on‐off” upconverting signals for readout. However, each UCNP contains thousands of emitting center ions and most of them are beyond the FRET critical distance, which hinders the fluorescence energy transfer efficiency, resulting in a low signal‐to‐background ratio (SBR). Herein, a new design is presented in which the energy of UCNPs is transferred to the o‐quinones on their surface via the photoinduced electron transfer (PET) mechanism. In this system, the quenching efficiency of UCNPs' fluorescence can be up to 94.73%, providing a high SBR. The performance of the PET‐based design is systematically testified, and the high‐sensitivity detection of disease biomarkers (tyrosinase and alkaline phosphatase) is demonstrated. Moreover, this UCNP‐PET platform is also capable of sensing the simulant of nerve agent sarin. This work will pave new ways to the design of UCNP‐based platforms toward bioanalytical applications.

Carbon quantum dot-gold nanocluster nanosatellite for ratiometric fluorescence probe and imaging for hydrogen peroxide in living cells

Developing probes for sensitive H2O2 detection is critical for understanding its related physiological and pathological processes. In this study, novel carbon quantum dot-gold nanocluster (CQD-AuNC) nanosatellite with high fluorescence energy transfer efficiency (∼40%) and dual-emission fluorescent properties have been exploited for fluorescence ratiometric sensing of H2O2. The sensing mechanism was based on the fact that H2O2 can quench the red emission of AuNCs. Under the optimal conditions, the CQD-AuNC nanosatellite probe was sensitive to H2O2 in the range from 5.0nM to 80nM with a limit of detection of 2.9nM. The practicality of the proposed CQD-AuNC nanosatellite probe was validated by imaging H2O2 in living cells.

FRET study of G-quadruplex forming fluorescent oligonucleotide probes at the lipid monolayer interface

Spectral properties and G-quadruplex folding ability of fluorescent oligonucleotide probes at the cationic dioctadecyldimethylammonium bromide (DODAB) monolayer interface are reported. Two oligonucleotides, a 19-mer bearing thrombin binding aptamer sequence and a 21-mer with human telomeric sequence, were end-labeled with fluorescent groups (FAM and TAMRA) to give FRET probes F19T and F21T, respectively. The probes exhibited abilities to fold into a quadruplex structure and to bind metal cations (Na+ and K+). Fluorescence spectra of G-quadruplex FRET probes at the monolayer interface are reported for the first time. Investigations included film balance measurements (π-A isotherms) and fluorescence spectra recording using a fiber optic accessory interfaced with a spectrofluorimeter. The effect of the presence of DODAB monolayer, metal cations and the surface pressure of monolayer on spectral behavior of FRET probes were examined. Adsorption of probe at the cationic monolayer interface resulted in the FRET signal enhancement even in the absence of metal cations. Variation in the monolayer surface pressure exerted rather modest effect on the spectral properties of probes. The fluorescence energy transfer efficiency of monolayer adsorbed probes increased significantly in the presence of sodium or potassium ion in subphase, which indicated that the probes retained their cation binding properties when adsorbed at the monolayer interface.

Effect of Cholesterol Anchoring Group on the Properties of G-Quadruplex-Based FRET Probes for Potassium Ion

In this study we report on the spectral properties and G-quadruplex folding ability of fluorescent oligonucleotide probes modified by the attachment of a cholesterol moiety. These probes were designed and studied in order to verify their potential as potassium-sensing devices that can be incorporated into the cellular membrane. The 19-meric guanine-rich deoxyoligonucleotide was labeled with reporter fluorescent FRET groups (FAM and TAMRA) and a cholesterol anchor was attached using different approaches. The probes exhibited abilities to fold into a quadruplex structure and to bind metal cations (Na+ and K+). In an unbound state, both termini of the oligonucleotide are separated, thus fluorophores do not interact with each other and the probe exhibits an unperturbed fluorescence spectrum. In the presence of K+, the quadruplex structure is developed such that it enables fluorophores to be arranged in close proximity, causing generation of a different fluorescence spectrum (FRET signal). Folding properties of probes and their spectral behavior were examined by recording the UV-Vis, fluorescence emission, and excitation spectra (FRET efficiency), and the temperature stability of G-quadruplex structures adopted by probes (melting profiles). Fluorescence energy transfer efficiency increased with increases in sodium or potassium ion concentrations in an aqueous solution, which indicated that the probes retained their cation-binding properties and could be promising candidates for potassium sensing at the cell membrane interface.

Detailed microscopic unfolding pathways of an α-helix and a β-hairpin: direct observation and molecular dynamics.

We present a combined experimental and computational study of unfolding pathways of a model 21-residue α-helical heteropeptide (W1H5-21) and a 16-residue β-hairpin (GB41-56). Experimentally, we measured fluorescence energy transfer efficiency as a function of temperature, employing natural tryptophans as donors and dansylated lysines as acceptors. Secondary structural analysis was performed with circular dichroism and Fourier transform infrared spectroscopy. Our studies present markedly different unfolding pathways of the two elementary secondary structural elements. During thermal denaturation, the helical peptide exhibits an initial decrease in length, followed by an increase, while the hairpin undergoes a systematic increase in length. In the complementary computational part of the project, we performed microsecond length replica-exchange molecular dynamics simulations of the peptides in explicit solvent, yielding a detailed microscopic picture of the unfolding processes. For the α-helical peptide, we found a large heterogeneous population of intermediates that are primarily frayed single helices or helix-turn-helix motifs. Unfolding starts at the termini and proceeds through a stable helical region in the interior of the peptide but shifted off-center toward the C-terminus. The simulations explain the experimentally observed non-monotonic variation of helix length with temperature as due primarily to the presence of frayed-end single-helix intermediate structures. For the β-hairpin peptide, our simulations indicate that folding is initiated at the turn, followed by formation of the hairpin in zipper-like fashion, with Cα···Cα contacts propagating from the turn to termini and hairpin hydrogen bonds forming in parallel with these contacts. In the early stages of hairpin formation, the hydrophobic side-chain contacts are only partly populated. Intermediate structures with low numbers of β-hairpin hydrogen bonds have very low populations. This is in accord with the "broken zipper" model of Scheraga. The monotonic increase in length with temperature may be explained by the zipper-like breaking of the hairpin hydrogen bonds and backbone contacts.

Squeezing at Entrance of Proton Transport Pathway in Proton-translocating Pyrophosphatase upon Substrate Binding

Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding.

Multidonor Deep-UV FRET Study of Protein–Ligand Binding and Its Potential to Obtain Structure Information

Fluorescence resonance energy transfer (FRET) using biotinylated β-galactosidase (βGAL) as a donor and Alexa Fluor 350 (AF350) labeled avidin as an acceptor has been investigated by means of steady-state fluorescence and time-resolved fluorescence spectroscopy. The donors are readily paired with acceptors through the well-established binding affinity of biotin and avidin. The fluorescence energy transfer efficiency was determined by the donor fluorescence emission and lifetime changes in the presence and absence of acceptor. The theoretical energy transfer efficiency and theoretical average distance between donor and acceptor after noncovalent binding was calculated by taking the distribution of tryptophan residues in βGAL and avidin as well as the location of AF350 in avidin into account, which agree with the experimental data. It is shown how information of the location of the acceptor can be obtained. Further, the fluorescence intensity image of AF350 on a biotinylated βGAL-coated quartz surface through UV FRET has been recorded using deep UV laser-based fluorescence lifetime microscopy. The results demonstrate that (a) deep UV laser-based fluorescence lifetime microscopy is a simple and useful method to study UV FRET of proteins using intrinsic fluorescence, (b) structural information even in complex multidonor systems can be obtained, and (c) FRET signals can be obtained to detect binding events using the native fluorescence of proteins as multidonor systems.

Fluorescence energy transfer efficiency in labeled yeast cytochrome c: a rapid screen for ion biocompatibility in aqueous ionic liquids

A fluorescence energy transfer "de-quenching" assay was implemented to follow the equilibrium unfolding behaviour of site-specific tetramethylrhodamine-labelled yeast cytochrome c in aqueous ionic liquid solutions; additionally, this approach offers the prospect of naked eye screening for biocompatible ion combinations in hydrated ionic liquids.

Three-dimensional molecular modeling with single molecule FRET

Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.

Regionalization of Plasma Membrane-Bound Flavoproteins of Cerebellar Granule Neurons in Culture by Fluorescence Energy Transfer Imaging

Flavoproteins are components of plasma membrane redox chains, which have been suggested to play major roles in neuronal activity and survival. We found that the red/orange autofluorescence of mature primary cultures of cerebellar granule neurons (8-9 days in vitro) was largely quenched by millimolar concentrations of dithionite added to the extracellular medium, and pointed out that nearly 50% of this autofluorescence was due to plasma membrane-bound flavoproteins. We report in this work that the lipophilic neuronal plasma membrane markers N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)-pyridinium dibromide (RH-414) and N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4-64) can form fluorescence energy transfer donor-acceptor pairs with flavoproteins with calculated R (0) values between 3.7 and 4.2 nm. The quantification of the efficiency of fluorescence energy transfer with different concentrations of acceptor dyes has been worked out with re-suspended neurons. Using quantitative images of the neurons in culture, acquired with a CCD camera attached to an epifluorescence microscope, regionalization of the plasma membrane-bound flavoproteins of cerebellar granule neurons has been achieved from the quenching by dithionite of the fluorescence of the acceptor dye. The results unraveled that plasma membrane-bound flavoproteins are largely enriched in interneuronal contact sites forming clusters of 0.5-1 microm diameter size, which appears largely regionalized in the neuron's cell body.

DNA photonics

Abstract Short DNA duplexes can be stabilized by the presence of organic chromophores, which serve as hairpin linkers or end-capping groups. Capped hairpins possessing one or more base pairs form stable folded structures in aqueous solution. Increasing the number of base pairs separating the two chromophores increases both the distance between the two chromophores and the dihedral angle between their electronic transition dipoles. Thus, duplex DNA can serve as a helical scaffold for the study of electronic interactions between two chromophores. Three types of electronic interaction have been investigated: (a) exciton coupling (EC) between two identical chromophores, as probed by exciton-coupled circular dichroism (EC-CD); (b) fluorescence resonance energy transfer (FRET) between a fluorescent donor and acceptor; and (c) photoinduced electron transfer (PET) between an electron donor and acceptor. EC and the efficiency of fluorescence energy transfer are dependent upon both the distance and dihedral angle separating the two chromophores. Electron transfer occurs via both single-step superexchange and bridge-mediated hopping mechanisms, neither of which displays angular dependence. The competition between these mechanisms is dependent upon both the energetics of hole injection into the base-pair bridge and the distance between the donor and acceptor chromophores, superexchange dominating at short distance and hole hopping at longer distances.

Decavanadate binding to a high affinity site near the myosin catalytic centre inhibits F-actin-stimulated myosin ATPase activity.

Decameric vanadate (V(10)) inhibits the actin-stimulated myosin ATPase activity, noncompetitively with actin or with ATP upon interaction with a high-affinity binding site (K(i) = 0.27 +/- 0.05 microM) in myosin subfragment-1 (S1). The binding of V(10) to S1 can be monitored from titration with V(10) of the fluorescence of S1 labeled at Cys-707 and Cys-697 with N-iodo-acetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) or 5-(iodoacetamido) fluorescein, which showed the presence of only one V(10) binding site per monomer with a dissociation constant of 0.16-0.7 microM, indicating that S1 labeling with these dyes produced only a small distortion of the V(10) binding site. The large quenching of AEDANS-labeled S1 fluorescence produced by V(10) indicated that the V(10) binding site is close to Cys-697 and 707. Fluorescence studies demonstrated the following: (i) the binding of V(10) to S1 is not competitive either with actin or with ADP.V(1) or ADP.AlF(4); (ii) the affinity of V(10) for the complex S1/ADP.V(1) and S1/ADP.AlF(4) is 2- and 3-fold lower than for S1; and (iii) it is competitive with the S1 "back door" ligand P(1)P(5)-diadenosine pentaphosphate. A local conformational change in S1 upon binding of V(10) is supported by (i) a decrease of the efficiency of fluorescence energy transfer between eosin-labeled F-actin and fluorescein-labeled S1, and (ii) slower reassociation between S1 and F-actin after ATP hydrolysis. The results are consistent with binding of V(10) to the Walker A motif of ABC ATPases, which in S1 corresponds to conserved regions of the P-loop which form part of the phosphate tube.

Steady-state and time-resolved fluorescence studies of conformational changes induced by cyclic AMP and DNA binding to cyclic AMP receptor protein from Escherichia coli.

cAMP receptor protein (CRP), allosterically activated by cAMP, regulates the expression of several genes in Escherichia coli. As binding of cAMP leads to undefined conformational changes in CRP, we performed a steady-state and time-resolved fluorescence study to show how the binding of the ligand influences the structure and dynamics of the protein. We used CRP mutants containing a single tryptophan residue at position 85 or 13, and fluorescently labeled with 1,5-I-AEDANS attached to Cys178. Binding of cAMP in the CRP-(cAMP)2 complex leads to changes in the Trp13 microenvironment, whereas its binding in the CRP-(cAMP)4 complex alters the surroundings of Trp85. Time-resolved anisotropy measurements indicated that cAMP binding in the CRP-(cAMP)2 complex led to a substantial increase in the rotational mobility of the Trp13 residue. Measurement of fluorescence energy transfer (FRET) between labeled Cys178 and Trp85 showed that the binding of cAMP in the CRP-(cAMP)2 complex caused a substantial increase in FRET efficiency. This indicates a decrease in the distance between the two domains of the protein from 26.6 A in apo-CRP to 18.7 A in the CRP-(cAMP)2 complex. The binding of cAMP in the CRP-(cAMP)4 complex resulted in only a very small increase in FRET efficiency. The average distance between the two domains in CRP-DNA complexes, possessing lac, gal or ICAP sequences, shows an increase, as evidenced by the increase in the average distance between Cys178 and Trp85 to approximately 20 A. The spectral changes observed provide new structural information about the cAMP-induced allosteric activation of the protein.

Synthesis and properties of oligonucleotide duplexes containing donor and acceptor fluorophores at 2′-positions

A method for introduction of dimethylamionapthamide and fluorescein labels as a fluorescence energy donor and acceptor pair into 2′-positions of DNA duplexes has been described. It has been shown that the attachment of these bulky fluorophores to the sugar 2′-position at the terminal fraying end of each oligonucleotide strand does not alter the normal thermal stability and global conformation of the DNA duplexes. A clear dependence of fluorescence energy transfer efficiency on the number of nucleotides in DNA has been observed, suggesting that the present donor-acceptor pair may be useful for FRET indicator of DNA.

Estimation of the distance change between cysteine-457 and the nucleotide binding site when sodium pump changes conformation from E1 to E2 by fluorescence energy transfer measurements.

The first indication of the size of a conformational change implicated in ion transport by sodium pump has been obtained by measuring the change in efficiency of fluorescence energy transfer between two specific locations on the alpha-subunit. The donor (5'-(iodoacetamido)fluorescein) attaches covalently to cysteine-457, and the acceptor (2'(or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate) binds reversibly to the active site. The acceptor binds nearly 2 orders of magnitude tighter to the Na+ than to the K+ conformation of the enzyme and quenches donor fluorescence more efficiently in the Na+ than in the K+ conformation. The estimated distance between donor and acceptor, assuming random orientation of their emission and absorption dipoles, increases 2.9 +/- 0.6 A when the enzyme changes from the Na+ to the K+ conformation. Stopped-flow measurements of the change in fluorescence energy transfer efficiency with time when the doubly-labeled pump is mixed with Na+ or K+ demonstrate that the donor/acceptor pair reports the change between the E1 and E2 conformations of unphosphorylated enzyme. The observed first-order rate constant for the change in energy transfer efficiency depends sigmoidally on [K+] and inversely on [Na+], and both rate and amplitude data for the change in energy transfer efficiency can be fit with the same values of the rate and ion-dissociation constants as published data for the conformational change between E1 and E2 obtained by singly labeling the enzyme with fluorophores that report changes in protein microenvironment. The prerequisite for successfully measuring the distance change and equating the protein rearrangement with a step in the catalysis-transport cycle is that the donor by itself does not report the conformational change.

Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics

We describe practical aspects of photobleaching fluorescence energy transfer measurements on individual living cells. The method introduced by T. M. Jovin and co-workers (see, most recently, Kubitscheck et al. 1993. Biophys. J. 64:110) is based on the reduced rate of irreversible photobleaching of donor fluorophores when acceptor fluorophores are present. Measuring differences in donor photobleaching rates on cells labeled with donor only (fluorescein isothiocyanate-conjugated proteins) and with both donor and acceptor (tetramethylrhodamine-conjugated proteins) allows calculation of the fluorescence energy transfer efficiency. We assess possible methods of data analysis in light of the underlying processes of photobleaching and energy transfer and suggest optimum strategies for this purpose. Single murine B lymphocytes binding various ratios of donor and acceptor conjugates of tetravalent concanavalin A (Con A) and divalent succinyl Con A were examined for interlectin energy transfer by these methods. For Con A, a maximum transfer efficiency of 0.49 +/- 0.02 was observed. Under similar conditions flow cytometric measurements of donor quenching yielded a value of 0.54 +/- 0.03. For succinyl Con A, the maximum transfer efficiency was 0.36. To provide concrete examples of quantities arising in such energy transfer determinations, we present examples of individual cell data and kinetic analyses, population rate constant distributions, and error estimates for the various quantities involved.

Reactivity of lysyl residues on the (calcium-magnesium)-ATPase to 7-amino-4-methylcoumarin-3-acetic acid succinimidyl ester

The (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum was labeled with the succinimidyl ester of 7-amino-4-methylcoumarin-3-acetic (AMCA). Although a large number of residues were labeled, it was found that Lys-492 was labeled preferentially at pH values between 6 and 8, consistent with an unusual environment for this residue. Labeling was reduced in the presence of ATP, suggesting that Lys-492 is in or near the ATP binding site of the ATPase. Other identified residues labeled by AMCA were Lys-35, Lys-135, Lys-218, Lys-371, and Lys-605. It is suggested that these represent surface-exposed lysyl residues. Lys-515, labeled by fluorescein isothiocyanate (FITC), was not labeled by AMCA. Labeling with AMCA at pH 6.0 has no effect on ATPase activity, suggesting that Lys-492 is not essential for activity. The fluorescence of AMCA-labeled ATPase did not change on addition of either ATP in the presence of Ca2+ or Pi in the absence of Ca2+, suggesting that Lys-492 was not affected by any major conformational changes on the ATPase. The efficiency of fluorescence energy transfer between AMCA and FITC labels on the ATPase was unaffected by binding Ca2+ or vanadate, arguing against any large-scale movement of the cytoplasmic domains of the ATPase.

Electrostatic potential around actin.

We presume that tension of contracting muscle originates from electrostatic force experienced by actin and myosin. We suppose that a high-energy state of myosin-ADP-Pi interacts with actin, transferring the stored energy to actin, and that the actin excited in this way develops around itself electric field which exerts sliding force against charged myosin heads. To explore the idea, first we conjectured how electric charges on actin produce electric field in the axial direction, and second we experimentally examined electrostatic circumstances around actin in solution and in muscle fibers by optimizing diffusion-enhanced fluorescence energy transfer. In the experiments, Tb ion, which has a long excited-state lifetime, was used as donor. To introduce Tb to actin, Tb-DTPA-phalloin and Tb-DTPA-maleimide were synthesized. As acceptors with electric charges (Za = -3 to +2), rhodamine B that was conjugated with various amino acids or their derivatives was used. The fluorescence energy transfer efficiency (ET) was estimated from the shortening in the lifetime of Tb. The electrostatic circumstances around actin were inferred from the ET-Za relation. When Tb was introduced at Cys-374 of actin, the Tb-site was found in negative electric potential. S-1 binding to the labeled actin neutralized the electric potential almost completely. Tb-DTPA-phalloin bound to actin seemed to reside in the vicinity of tryptophan residue(s). Electric potential around the phalloin site was negative. S-1 binding to the actin slightly reduced the negativity. In glycerinated fibers in the rigor state, the phalloin site was again found in negative potential. When fibers were transferred from an ADP-rigor solution to an active solution, the negative electric potential was neutralized to some extent. The direction of this change could not be explained by detachment of crossbridges from actin, since the detachment should have given an opposite direction of changes in the electric potential. Thus, this observation may indicate that electric potential characteristic of the active state occurs at actin surfaces.