Intramolecular FRET Research Articles

A more flexible long-chain bis(salamo)-like fluorescent probe based on FRET and ICT effects for the identification of aluminum ion in the environment and plants

A more flexible long-chain bis(salamo)-like fluorescent probe DS was designed and synthesized. The unique coupling systems of FRET and ICT effects were designed for DS, which was demonstrated to be capable of detecting aluminum ion through the FRET effect, changing the fluorescence of the solution from green to bright blue (55 nm blue-shift) with good selectivity and fast response, and detecting Al3+ over a wide pH range. The recognition mechanism was determined based on spectral characterization, in the sense that the coordination of Al3+ with the probe DS molecule turned on the intramolecular FRET and ICT processes. ESP analysis and DFT calculations validated the binding sites and fluorescence recognition mechanism. Finally, good results have been achieved in practical applications. The test paper experiment confirmed that DS can be prepared into portable Al3+ test paper, the fluorescence imaging experiment of mung bean sprouts showed that DS can be used for the fluorescence imaging of Al3+ in plants, and the environmental water samples test proved that various ions in the actual water samples hardly interfere with the identification of Al3+, and it is expected to be applied in the environmental testing and other fields.

Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

Complexes of charged disordered polyelectrolyte-like proteins: From thermodynamics to stuctural plasticity.

The highly charged IDPs ProTα and H1 (−44 and +53, respectively) form a high-affinity, yet disordered complex and thus define a limiting case in the spectrum of disorder in IDP complexes [1-3]. Temperature-dependent single-molecule FRET (smFRET) experiments and isothermal titration calorimetry (ITC) reveal ProTα-H1 complexation to be enthalpically unfavorable. Using smFRET, we also identify stoichiometrically defined ternary complexes between ProTα and H1 at equilibrium and quantitate their salt-dependent stabilities, allowing us to accurately analyze ensemble experiments such as ITC performed with micromolar protein concentrations, where ternary complex formation is pronounced. Salt-dependent measurements show that counterion release entropy is a dominant driving force of complexation. An analytical polymer theory for polyelectrolyte complexation that explicitly accounts for counterions explains the thermodynamic results and suggests that the positive binding enthalpy is dominated by the relative exothermicities of counterion condensation and interchain ion-pair formation. Despite its high affinity, the disordered complex shows remarkable plasticity and expands continuously with increasing salt concentration, as seen from inter- and intramolecular FRET using various ProTα and H1 labeling variants. However, the salt-dependent expansion is non-uniform, which can be explained by differences in charge densities in different ProTα and H1 segments. Our investigations provide a comprehensive framework for the unconventional behavior of polyelectrolyte-like biomolecular complexes.

Phospholamban inhibits the cardiac calcium pump by reversing allosteric effects of ATP on calcium affinity.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+ ATPase (SERCA) in cardiac muscle. PLB reduces the apparent Ca2+ sensitivity of SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, and the pump is not cycling. One prevailing explanation for these seemingly conflicting results is PLB may slow the Ca2+ binding step in the SERCA enzymatic cycle, altering the Ca2+ dependence of cycling without affecting true Ca2+ affinity. Alternatively, we speculated that equilibrium measurements of Ca2+ binding to non-cycling SERCA may overlook important allosteric effects of bound nucleotide. Specifically, ATP is known to have an allosteric effect on SERCA that is distinct from its role as a catalytic substrate and source of energy. When ATP binds, it increases the affinity of SERCA for Ca2+ - exactly the opposite of the PLB effect. We speculated that PLB may inhibit SERCA by reversing pre-activation of SERCA Ca2+ affinity by ATP. To test this hypothesis, we used a 2-color SERCA biosensor expressed in microsomal membranes to measure Ca2+ binding to the pump reported by changes in intramolecular FRET. We quantified the Ca2+ affinity of non-cycling SERCA in the absence of ATP, then mimicked ATP binding while preventing enzymatic cycling using the non-hydrolyzable ATP-analog AMPPCP. Interestingly, nucleotide activation by AMPPCP increased the Ca2+ affinity of 2-color SERCA, but this effect was reversed by co-expression of PLB. PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results may reconcile the seemingly paradoxical effects of PLB. We propose that PLB reduces the true Ca2+ affinity of SERCA by reversing allosteric activation of the pump by ATP.

Rational Construction of a Mitochondria-Targeted Reversible Fluorescent Probe with Intramolecular FRET for Ratiometric Monitoring Sulfur Dioxide and Formaldehyde.

Sulfur dioxide (SO2) and formaldehyde (FA) are important species that maintain redox homeostasis in life and are closely related to many physiological and pathological processes. Therefore, it is of great significance to realize the reversible monitoring of them at the intracellular level. Here, we synthesized a reversible ratiometric fluorescent probe through a reasonable design, which can sensitively monitor SO2 derivatives and FA, and the detection limit can reach 0.16 μM. The probe can specifically target mitochondria and successfully monitor the fluctuations of SO2 and FA in living cells. It also works well in the detection of SO2 and FA in zebrafish. This high-performance probe is expected to find broad in vitro and in vivo applications.

Cardiac ryanodine receptor N-terminal region biosensors identify novel inhibitors via FRET-based high-throughput screening

The N-terminal region (NTR) of ryanodine receptor (RyR) channels is critical for the regulation of Ca2+ release during excitation–contraction (EC) coupling in muscle. The NTR hosts numerous mutations linked to skeletal (RyR1) and cardiac (RyR2) myopathies, highlighting its potential as a therapeutic target. Here, we constructed two biosensors by labeling the mouse RyR2 NTR at domains A, B, and C with FRET pairs. Using fluorescence lifetime (FLT) detection of intramolecular FRET signal, we developed high-throughput screening (HTS) assays with these biosensors to identify small-molecule RyR modulators. We then screened a small validation library and identified several hits. Hits with saturable FRET dose–response profiles and previously unreported effects on RyR were further tested using [3H]ryanodine binding to isolated sarcoplasmic reticulum vesicles to determine effects on intact RyR opening in its natural membrane. We identified three novel inhibitors of both RyR1 and RyR2 and two RyR1-selective inhibitors effective at nanomolar Ca2+. Two of these hits activated RyR1 only at micromolar Ca2+, highlighting them as potential enhancers of excitation–contraction coupling. To determine whether such hits can inhibit RyR leak in muscle, we further focused on one, an FDA-approved natural antibiotic, fusidic acid (FA). In skinned skeletal myofibers and permeabilized cardiomyocytes, FA inhibited RyR leak with no detrimental effect on skeletal myofiber excitation–contraction coupling. However, in intact cardiomyocytes, FA induced arrhythmogenic Ca2+ transients, a cautionary observation for a compound with an otherwise solid safety record. These results indicate that HTS campaigns using the NTR biosensor can identify compounds with therapeutic potential.

Modulation of Aggregation-Caused Quenching to Aggregation-Induced Emission: Finding a Biocompatible Polymeric Theranostics Platform for Cancer Therapy.

Dual intramolecular FRET polymers are synthesized via Suzuki coupling and their luminescence characteristics from aggregation-caused quenching (ACQ) to aggregation-induced emission (AIE) is modulated conveniently by adjusting the charged ratios. The finally obtained AIE polymer is further employed to construct doxorubicin loaded nanoparticles as a promising theranostics platform for cancer therapy.

FLTX2: A Novel Tamoxifen Derivative Endowed with Antiestrogenic, Fluorescent, and Photosensitizer Properties.

Tamoxifen is the most widely used selective modulator of estrogen receptors (SERM) and the first strategy as coadjuvant therapy for the treatment of estrogen-receptor (ER) positive breast cancer worldwide. In spite of such success, tamoxifen is not devoid of undesirable effects, the most life-threatening reported so far affecting uterine tissues. Indeed, tamoxifen treatment is discouraged in women under risk of uterine cancers. Recent molecular design efforts have endeavoured the development of tamoxifen derivatives with antiestrogen properties but lacking agonistic uterine tropism. One of this is FLTX2, formed by the covalent binding of tamoxifen as ER binding core, 7-nitrobenzofurazan (NBD) as the florescent dye, and Rose Bengal (RB) as source for reactive oxygen species. Our analyses demonstrate (1) FLTX2 is endowed with similar antiestrogen potency as tamoxifen and its predecessor FLTX1, (2) shows a strong absorption in the blue spectral range, associated to the NBD moiety, which efficiently transfers the excitation energy to RB through intramolecular FRET mechanism, (3) generates superoxide anions in a concentration- and irradiation time-dependent process, and (4) Induces concentration- and time-dependent MCF7 apoptotic cell death. These properties make FLTX2 a very promising candidate to lead a novel generation of SERMs with the endogenous capacity to promote breast tumour cell death in situ by photosensitization.

The C-terminal of the α1b-adreneroceptor is a key determinant for its structure integrity and biological functions.

The C-terminal of G protein-coupled receptors is now recognized as being important for G protein activation and signaling function. To detect the role of C-terminal tail in receptor activation, we used the α1b-AR, which has a long C-terminal of 164 amino acids. We constructed the intramolecular FRET sensors, in which the C-terminal was truncated to 10 (∆C-10), 20 (∆C-20), 30 (∆C-30), 50 (∆C-50), 70 (∆C-70), or 90 (∆C-90). The truncated mutants of ∆C-10, ∆C-20, or ∆C-30 cannot induce FRET signal changes and downstream ERK1/2 phosphorylation. However, the truncated mutants of ∆C-50, ∆C-70, or ∆C-90 induce significant FRET signal changes and downstream ERK1/2 phosphorylation, especially ∆C-90. This is particularly true in the case of the ∆C-90, ∆C-70, or ∆C-50 which retained the potential phosphorylation sites (Ser401, Ser404, Ser408, or Ser410). The ∆C-90 showed an increase in agonist-induced FRET signal changes and ERK1/2 phosphorylation in PKC- or endocytosis-dependent and EGFR-, src-, or β-arrestin2-independent.

High-Throughput FRET Screening in Living Cells Based on Lifetime Detection to Identify Small-Molecule Effectors of Serca

The development of an effective high-throughput screening (HTS) paradigm using FRET biosensors in living cells has enabled us to identify multiple classes of small-molecules that alter the activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). We aim to discover small-molecule activators that improve SERCA's function for the treatment of heart failure. We have validated an intramolecular SERCA FRET biosensor (red and green fluorescent proteins fused to distinct SERCA domains) by screening a small test library using novel plate readers (PRs) that detect the fluorescence lifetime (FLT) (Schaaf et al, 2017). Here we describe an FLT-PR that scans a 1536-well plate in ≤2.5 min. This instrument is equipped with multichannel FLT detection at two emission wavelengths, used to filter out fluorescent compounds that would be falsely identified as hits. False hits were further eliminated using FRET biosensors based on non-specific peptide linkers, and using spectral analysis. FRET biosensors were developed and tested based on two different isoforms of SERCA: the SERC2a cardiac-specific isoform and SERCA2b non-muscle isoform were used to discover compounds with specificity toward SERCA2a. Concentration-response studies were performed to determine potency of compounds. Hits were assessed by two separate functional assays to determine how they alter ATPase activity and calcium-uptake. The majority were found to be inhibitors, but some were activators, and some compounds affected the coupling of SERCA's ATPase and calcium transport activities Our future focus is to evaluate the structure-function relationships of the newly discovered SERCA activators, using medicinal chemistry to optimize drug-like properties. These new HTS methods are being applied to a wide range of drug targets.

A near infrared fluorescent probe for one-step detection of histone deacetylase activity based on an intramolecular FRET

It is urgent to implement ideal fluorophores to detect enzymatic activity of histone deacetylases (HDACs) because of their important roles in many cellular metabolisms such as transcription regulation and tumorigenesis. However, the emission wavelength of reported one-step fluorescent probes for the detection of HDACs is limited at visible region. In this paper, we construct a near infrared (NIR) fluorescent probe Lys-OBD for detecting the activity of sirtuin 1 (SIRT1), one of the most important HDACs, with NIR emission based on an intramolecular FRET effect. Lys-OBD contains a non-emissive O-aryl NBD group (OBD) and a NIR emissive Cy5 fluorophore. After reaction with SIRT1, non-fluorescence OBD in Lys-OBD was changed to green emissive nitrobenzoxadiazole (NBD) through a deacetylation reaction followed by a backbone rearrangement. An intramolecular FRET between the energy donor NBD and the energy acceptor Cy5 was thus happened and the NIR emission of Cy5 could be detected. This FRET mechanism could further decrease the interference of high-concentration amino acid that are abound in living system in the process of SIRT1 detection. Thus, this probe can not only detect SIRT1 in vitro, but also image SIRT1 in live cells that high express SIRT1 with NIR fluorescence for the first time.

Developing a novel FRET assay, targeting the binding between Antizyme-AZIN

Antizyme inhibitor (AZIN) stimulates cell proliferation by binding to and sequestering the cell cycle suppressor antizyme. Despite the important role of the antizyme-AZIN protein-protein interaction (PPI) in cell cycle regulation, there are no assays for directly measuring the binding of AZIN to antizyme that are amenable to high throughput screening. To address this problem, we developed and validated a novel antizyme-AZIN intramolecular FRET sensor using clover and mRuby2 fluorescent proteins. By introducing alanine mutations in the AZIN protein, we used this sensor to probe the PPI for key residues governing the binding interaction. We found that like many PPIs, the energy of the antizyme-AZIN binding interaction is distributed across many amino acid residues; mutation of individual residues did not have a significant effect on disrupting the PPI. We also examined the interaction between Clover-AZIN and antizyme-mRuby2 in cells. Evidence of a direct interaction between Clover-AZIN and antizyme-mRuby2 was observed within cells, validating the use of this FRET sensor for probing intracellular antizyme-AZIN PPI. In conclusion, we have developed and optimized a FRET sensor which can be adapted for high throughput screening of either in vitro or intracellular activity.

Light-harvesting porphyrazines to enable intramolecular singlet fission

A porphyrazine featuring complementary absorption to a pentacene dimer was chosen to fill the absorption gap of the latter in the range of 450 to 600 nm to realize panchromatic absorption through the visible region out to ca. 700 nm. Of even greater relevance is the quantitative intramolecular Förster resonance energy transfer (i-FRET) to funnel energy to the pentacene moieties, where efficient intramolecular singlet fission (i-SF) converts the singlet excited state into the corresponding triplet excited states. Remarkably, the triplet quantum yield either via direct excitation or via indirect i-FRET is up to 200% ± 20% in polar solvents.

Amyloid Beta Peptide Modulates the Signaling Property of Chemokine‐like Receptor 1

OBJECTIVEAmyloid beta peptides (Aβ) are the main constituents of the amyloid plaques found in the brains of Alzheimer patients. While Aβ is proinflammatory and contributes to the development of Alzheimer's disease, its signaling property remains unclear. The present study investigates how Aβ modulates signaling of one of its receptors, the chemokine‐like receptor 1 (CMKLR1, ChemR23).METHODSCells expressing CMKLR1 were incubated with Aβ42 before stimulation with another agonist of the CMKLR1, the C9 peptide. Calcium mobilization, cAMP inhibition and MAP kinase activation were measured. Intramolecular FRET was determined using CMKLR1 constructs containing an ECFP attached to the C‐terminus.RESULTSBinding of both Aβ42 and the C9 peptide induced CMKLR1 internalization, but only the Aβ42‐induced receptor internalization involved clathrin‐coated pits. Likewise, Aβ42 but not C9 stimulated β‐arrestin2 translocation to plasma membranes. A robust Ca2+ flux was observed following C9 stimulation, whereas Aβ42 was ineffective even at micromolar concentrations. Despite its low potency in calcium mobilization assay, Aβ42 was able to alter C9‐induced Ca2+ flux with potentiation at concentrations as low as 0.1 – 1 nM. However, at higher concentrations the effect was diminished. Similar effects were seen with the C9‐induced ERK phosphorylation but the dose curve was different from that of Ca2+ flux and cAMP inhibition, suggesting a reciprocal regulatory mechanism. Intramolecular FRET assay confirmed that Aβ42 modulates CMKLR1 rather than its downstream signaling pathways.CONCLUSIONThese findings suggest that long‐term exposure to Aβ42 may alter the signaling properties of its receptors, thereby influence the pathology of Alzheimer's disease. The reciprocal regulation of CMKLR1 by Aβ provides a novel mechanism by which a ligand dose‐dependently modulates the response of its receptor to another ligand.Support or Funding InformationSupported by grants SRG2015‐00047‐ICMS‐QRCM and MYRG2016‐00152‐ICMS‐QRCM from University of Macau and by grant 31470865 from NSFC.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fluorescent Dye Dynamics Attached to EF-Tu and their Effect on a Hetero-FRET System

Elongation Factor-Tu (EF-Tu) is an essential protein involved in the elongation cycle of the protein synthesis by delivering aminoacylated tRNAs to the ribosomal A-site. The active form of the protein has GTP bound to the G-domain. The G-domain along with two additional domains undergoes significant conformational transition to GDP-bound state, following GTP hydrolysis and Pi release. We developed an intra-molecular hetero-FRET system using Elongation Factor-Tu variants to study the details of these structural rearrangements in real time. This intra-molecular FRET system will also allow for the elucidation of EF-Tu's dynamic transitions during interactions with components of the bacterial translation machinery. Here we report molecular dynamics simulation of the protein with covalently linked to the FRET-dye pair and compare the results with equilibrium and rapid kinetics fluorescence experiments. The flexibility of the dye pair and their orientations were analysed and related to FRET parameters. Our finding suggests that it is critical for the selection of dyes and their location on the molecular surface of the protein to assess their flexibility in the context of the respective protein.

Color-tunable AIE-active conjugated polymer nanoparticles as drug carriers for self-indicating cancer therapy via intramolecular FRET mechanism

In this paper, two novel AIE-active conjugated polymers were synthesized by Pd-catalyzed Suzuki coupling polymerization reaction.

A New Compound-ensemble Strategy for Validating Cancer Drug Targets Confirms that Inhibiting Glycolysis Blocks Leukemic Cell Proliferation.

Cancer drug discovery programs fail too often. One reason may be inadequate small molecule support at the target validation stage as the tool compounds used often have multiple effects. Aerobic glycolysis - the Warburg effect - has reemerged as a potential cancer target of interest, but small molecule evidence is weak. We developed a new assay based on expression of an intramolecular FRET sensor for ATP in K562 cells and used it to screen a library for metabolically-active compounds. Seven inhibitors of oxidative phosphorylation-dependent ATP production were no more antiproliferative than the overall compound set, while ten novel glycolysis inhibitors were in the top third of most effective compounds. This is highly unlikely unless inhibiting glycolysis effectively blocks proliferation. Our results point to a new approach to providing small molecule support for potential drug targets that could be applied prospectively and could help reduce the failure rate of drug development efforts.

Abstract B020: Glycolysis inhibitors are antiproliferative in leukemic cells

Abstract The Warburg effect has recently reemerged as an exciting anticancer target. However, until relatively recently, the only inhibitors available were unspecific agents like 2-deoxyglucose (2-DG) and 3-bromopyruvate, which have effects apart from glycolysis inhibition that could play important roles in inhibiting growth. While more recent screens have identified blockers of specific glycolytic enzymes, questions about the role of glycolysis inhibition in their effects on proliferation–when those have even been reported–remain. Taken together with the fact that knockouts or knockdowns do not necessarily provide evidence that enzymatic inhibition of a target will produce therapeutic effects, the evidence that targeting glycolysis could be an effective antiproliferative strategy remains weak. We suspected that examining the effects of a panel of structurally and mechanistically diverse glycolysis-inhibiting compounds rather than focusing on a single compound would be informative. Any one compound that inhibits glycolysis could reduce proliferation via off-target effects. However, if inhibiting glycolysis is indeed a good way to prevent cell growth, then if we identify a sufficient number of glycolysis inhibitors and all are antiproliferative, it could provide strong support for the idea that effects on glycolysis are responsible for effects on proliferation. In contrast, if even one effective glycolysis inhibitor were to fail to block proliferation, it would falsify the mechanism. We screened the NCI’s Mechanistic Set III using a novel multiread assay based on an intramolecular FRET sensor for ATP expressed stably in K562 cells, which can maintain ATP levels via glycolysis. We identified 7 inhibitors of oxidative phosphorylation-dependent ATP production and 10 glycolysis inhibitors. We drew upon the NCI's measurements of the compound set's effects on proliferation in K562 cells, and found that the inhibitors of oxidative phosphorylation-dependent ATP production were no more effective at inhibiting growth than the overall compound collection. Some had no effect on growth, suggesting that this is pathway is not a valid antiproliferative target in K562 cells. By contrast, all of the glycolysis inhibitors were in the top third of most effective antiproliferative compounds. The chances of obtaining 10 compounds as effective at blocking proliferation from the library solely by chance, estimated using the binomial distribution, are less than one in 500,000. This provides strong support for the idea that inhibiting glycolysis is likely an effective means of blocking proliferation of K562 cells, and points to a strategy that can be used to provide small-molecule support for pursuing specific glycolytic enzymes or other cellular targets for cancer therapies. Citation Format: Ziyan Zhao, Adam Zweifach. Glycolysis inhibitors are antiproliferative in leukemic cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B020.

Cdc42 activation couples fluid shear stress to apical endocytosis in proximal tubule cells.

Cells lining the kidney proximal tubule (PT) respond to acute changes in glomerular filtration rate and the accompanying fluid shear stress (FSS) to regulate reabsorption of ions, glucose, and other filtered molecules and maintain glomerulotubular balance. Recently, we discovered that exposure of PT cells to FSS also stimulates an increase in apical endocytic capacity (Raghavan et al. PNAS, 111:8506–8511, 2014). We found that FSS triggered an increase in intracellular Ca2+ concentration ([Ca2+]i) that required release of extracellular ATP and the presence of primary cilia. In this study, we elucidate steps downstream of the increase in [Ca2+]i that link FSS‐induced calcium increase to increased apical endocytic capacity. Using an intramolecular FRET probe, we show that activation of Cdc42 is a necessary step in the FSS‐stimulated apical endocytosis cascade. Cdc42 activation requires the primary cilia and the FSS‐mediated increase in [Ca2+]i. Moreover, Cdc42 activity and FSS‐stimulated endocytosis are coordinately modulated by activators and inhibitors of calmodulin. Together, these data suggest a mechanism by which PT cell exposure to FSS is translated into enhanced endocytic uptake of filtered molecules.

Conformational transition of polystyrene in good solvents studied by fluorescence spectroscopy

In this paper, the conformational transition of polystyrene (PS) in good solvents (i.e., dichloroethane and decalin) is studied by intramolecular fluorescence resonance energy transfer (FRET, NRET). The characteristics of FRET and PS excimer fluorescence spectroscopy in the analysis of polymer solution are systematically described. We find that FRET is prior to PS excimer fluorescence spectroscopy due to the superior sensitivity and better spectroscopic signal resolution. Based on the FRET results, it can be concluded that the chain conformation almost keeps unchanged with the increase of polymer concentration before the concentration reaches the critical overlap concentration ( C *). This spectroscopic observation excludes the existence of dynamic contact transition. In the expected C * regime, a sharp break point is observed for the fluorescence intensity versus the increase of the concentration, which is in exactly agreement with the theoretical value of C *. Additionally, the polymer chain undergoes severe collapse when the concentration reaches the semidilute regime and increases continuously. This work demonstrates that intramolecular FRET is a robust technique to sensitively characterize C *. C * could be used as a threshold to describe the conformational transition of polymer in good solvents.