Förster Resonance Energy Transfer Probes Research Articles

Using Förster Resonance Energy Transfer (FRET) to Understand the Ubiquitination Landscape.

Förster resonance energy transfer (FRET) is a fluorescence technique that allows quantitative measurement of protein interactions, kinetics and dynamics. This review covers the use of FRET to study the structures and mechanisms of ubiquitination and related proteins. We survey FRET assays that have been developed where donor and acceptor fluorophores are placed on E1, E2 or E3 enzymes and ubiquitin (Ub) to monitor steady-state and real-time transfer of Ub through the ubiquitination cascade. Specialized FRET probes placed on Ub and Ub-like proteins have been developed to monitor Ub removal by deubiquitinating enzymes (DUBs) that result in a loss of a FRET signal upon cleavage of the FRET probes. FRET has also been used to understand conformational changes in large complexes such as multimeric E3 ligases and the proteasome, frequently using sophisticated single molecule methods. Overall, FRET is a powerful tool to help unravel the intricacies of the complex ubiquitination system.

FRETpredict: a Python package for FRET efficiency predictions using rotamer libraries

Förster resonance energy transfer (FRET) is a widely-used and versatile technique for the structural characterization of biomolecules. Here, we introduce FRETpredict, an easy-to-use Python software to predict FRET efficiencies from ensembles of protein conformations. FRETpredict uses a rotamer library approach to describe the FRET probes covalently bound to the protein. The software efficiently and flexibly operates on large conformational ensembles such as those generated by molecular dynamics simulations to facilitate the validation or refinement of molecular models and the interpretation of experimental data. We provide access to rotamer libraries for many commonly used dyes and linkers and describe a general methodology to generate new rotamer libraries for FRET probes. We demonstrate the performance and accuracy of the software for different types of systems: a rigid peptide (polyproline 11), an intrinsically disordered protein (ACTR), and three folded proteins (HiSiaP, SBD2, and MalE). FRETpredict is open source (GPLv3) and is available at github.com/KULL-Centre/FRETpredict and as a Python PyPI package at pypi.org/project/FRETpredict.

Specific FRET Probes Sensitive to Chitosan-Based Polymeric Micelles Formation, Drug-Loading, and Fine Structural Features.

Förster resonance energy transfer (FRET) probes are a promising tool for studying numerous biochemical processes. In this paper, we show the application of the FRET phenomenon to observe the micelle formation from surfactants, micelles self-assembling from chitosan grafted with fatty acid (oleic-OA, or lipoic-LA), cross-linking of SH groups in the micelle's core, and inclusion and release of the model drug cargo from the micelles. Using the carbodiimide approach, amphiphilic chitosan-based polymers with (1) SH groups, (2) crosslinked with S-S between polymer chains, and (3) without SH and S-S groups were synthesized, followed by characterization by FTIR and NMR spectroscopy. Two pairs of fluorophores were investigated: 4-methylumbelliferon-trimethylammoniocinnamate-rhodamine (MUTMAC-R6G) and fluorescein isothiocyanate-rhodamine (FITC-R6G). While FITC-R6G has been described before as an FRET-producing pair, for MUTMAC-R6G, this has not been described. R6G, in addition to being an acceptor fluorophore, also serves as a model cytostatic drug in drug-release experiments. As one could expect, in aqueous solution, FRET effect was poor, but when exposed to the micelles, both MUTMAC-R6G and FITC-R6G yielded a pronounced FRET effect. Most likely, the formation of micelles is accompanied by the forced convergence of fluorophores in the hydrophobic micelle core by a donor-to-acceptor distance (r) significantly closer than in the aqueous buffer solution, which was reflected in the increase in the FRET efficiency (E). Therefore, r(E) could be used as analytical signal of the micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC), yielding values in good agreement with the literature for similar systems. We found that the r-function provides analytically valuable information about the nature and mechanism of micelle formation. S-S crosslinking between polymer chains makes the micelle more compact and stable in the normal physiological conditions, but loosens in the glutathione-rich tumor microenvironment, which is considered as an efficient approach in targeted drug delivery. Indeed, we found that R6G, as a model cytostatic agent, is released from micelles with initial rate of 5%/h in a normal tissue microenvironment, but in a tumor microenvironment model (10 mM glutathione), the release of R6G from S-S stitched polymeric micelles increased up to 24%/h. Drug-loading capacity differed substantially: from 75-80% for nonstitched polymeric micelles to ~90% for S-S stitched micelles. Therefore, appropriate FRET probes can provide comprehensive information about the micellar system, thus helping to fine-tune the drug delivery system.

Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles

Fluorescence lifetime imaging microscopy (FLIM) is a popular modality to create additional contrast in fluorescence images. By carefully analyzing pixel-based nanosecond lifetime patterns, FLIM allows studying complex molecular populations. At the single-molecule or single-particle level, however, image series often suffer from low signal intensities per pixel, rendering it difficult to quantitatively disentangle different lifetime species, such as during Förster resonance energy transfer (FRET) analysis in the presence of a significant donor-only fraction. In this article we investigate whether an object localization strategy and the phasor approach to FLIM have beneficial effects when carrying out FRET analyses of single particles. Using simulations, we first showed that an average of ∼300 photons, spread over the different pixels encompassing single fluorescing particles and without background, is enough to determine a correct phasor signature (SD < 5% for a 4-ns lifetime). For immobilized single- or double-labeled dsDNA molecules, we next validated that particle-based phasor-FLIM-FRET readily allows estimating fluorescence lifetimes and FRET from single molecules. Thirdly, we applied particle-based phasor-FLIM-FRET to investigate protein-protein interactions in subdiffraction HIV-1 viral particles. To do this, we first quantitatively compared the fluorescence brightness, lifetime, and photostability of different popular fluorescent protein-based FRET probes when genetically fused to the HIV-1 integrase enzyme in viral particles, and conclude that eGFP, mTurquoise2, and mScarlet perform best. Finally, for viral particles coexpressing FRET-donor/acceptor-labeled IN, we determined the absolute FRET efficiency of IN oligomers. Available in a convenient open-source graphical user interface, we believe that particle-based phasor-FLIM-FRET is a promising tool to provide detailed insights in samples suffering from low overall signal intensities.

Two-step structural changes in M3 muscarinic receptor activation rely on the coupled Gq protein cycle

G protein-coupled receptors (GPCRs) regulate diverse intracellular signaling pathways through the activation of heterotrimeric G proteins. However, the effects of the sequential activation–deactivation cycle of G protein on the conformational changes of GPCRs remains unknown. By developing a Förster resonance energy transfer (FRET) tool for human M3 muscarinic receptor (hM3R), we find that a single-receptor FRET probe can display the consecutive structural conversion of a receptor by G protein cycle. Our results reveal that the G protein activation evokes a two-step change in the hM3R structure, including the fast step mediated by Gq protein binding and the subsequent slower step mediated by the physical separation of the Gαq and Gβγ subunits. We also find that the separated Gαq-GTP forms a stable complex with the ligand-activated hM3R and phospholipase Cβ. In sum, the present study uncovers the real-time conformational dynamics of innate hM3R during the downstream Gq protein cycle.

Development of Selective ADAMTS-5 Peptide Substrates to Monitor Proteinase Activity.

The dysregulation of proteinase activity is a hallmark of osteoarthritis (OA), a disease characterized by progressive degradation of articular cartilage by catabolic proteinases such as a disintegrin and metalloproteinase with thrombospondin type I motifs-5 (ADAMTS-5). The ability to detect such activity sensitively would aid disease diagnosis and the evaluation of targeted therapies. Förster resonance energy transfer (FRET) peptide substrates can detect and monitor disease-related proteinase activity. To date, FRET probes for detecting ADAMTS-5 activity are nonselective and relatively insensitive. We describe the development of rapidly cleaved and highly selective ADAMTS-5 FRET peptide substrates through in silico docking and combinatorial chemistry. The lead substrates 3 and 26 showed higher overall cleavage rates (∼3-4-fold) and catalytic efficiencies (∼1.5-2-fold) compared to the best current ADAMTS-5 substrate ortho-aminobenzoyl(Abz)-TESE↓SRGAIY-N-3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl(Dpa)-KK-NH2. They exhibited high selectivity for ADAMTS-5 over ADAMTS-4 (∼13-16-fold), MMP-2 (∼8-10-fold), and MMP-9 (∼548-2561-fold) and detected low nanomolar concentrations of ADAMTS-5.

Two-step structural changes in M3 muscarinic receptor activation rely on the coupled G protein cycle.

G protein-coupled receptors (GPCRs) regulate diverse intracellular signaling pathways through the activation of heterotrimeric G proteins. However, the effects of the reversible activation-deactivation cycle of G protein on the conformational changes of GPCRs remains unknown. By developing a new Förster resonance energy transfer (FRET) tool for human M3 muscarinic acetylcholine receptor, we found that a single-receptor FRET probe can display the consecutive structural conversion of a receptor by G protein cycle. Our results revealed that the G protein activation evokes a two-step change in the hM3R structure, including the fast step mediated by Gq protein binding and the subsequent slower step mediated by the physical separation of the Gαq and Gβγ subunits. We also found that the separated Gαq-GTP forms a stable complex with the ligand-activated hM3R and phospholipase Cβ. In sum, the present study uncovered the real-time conformational dynamics of innate hM3R during the downstream Gq protein cycle.

Quantum Dot-Peptide Conjugates as Energy Transfer Probes for Sensing the Proteolytic Activity of Matrix Metalloproteinase-14.

We detail the assembly and characterization of quantum dot (QD)-dye conjugates constructed using a peptide bridge specifically designed to recognize and interact with a breast cancer biomarker─matrix metalloproteinase-14 (MMP-14). The assembled QD conjugates are then used as optically addressable probes, relying on Förster resonance energy transfer (FRET) interactions as a transduction mechanism to detect the activity of MMP-14 in solution phase. The QDs were first coated with dithiolane poly(ethylene glycol) (PEG) bearing a carboxyl group that allows coupling via amide bond formation with different dye-labeled peptides. The analytical capability of the conjugates is enabled by correlating changes in the FRET efficiency with the conjugate valence and/or QD-to-dye separation distance, triggered and modulated by enzymatic proteolysis of surface-tethered peptides. The FRET probe exhibits great sensitivity to enzyme digestion with sub-nanomolar limit of detection. We further analyze the proteolysis data within the framework of the Michaelis-Menten model, which considers the fact that surface-attached peptides have a slower diffusion coefficient than free peptides. This results in reduced collision frequency and lower catalytic efficiency, kcat/KM. Our results suggest that our conjugate design is promising, effective, and potentially useful for in vivo analysis.

Anti-Counterfeiting Inks Based on Förster Resonance Energy Transfer in Microcrystalline Cellulose-Grafted Poly(amidoamine) for Artificial Industries

Counterfeiting and inverse engineering of different artificial industries have significantly increased, which is a challenge for the originality of the crafts that are known as the cultural products of different nations. To prevent counterfeiting in the art market, it is needed to mark the original crafts with anti-counterfeiting inks using high technologies for their easy authentication. In this study, poly(amidoamine) (PAMAM) dendrimer-modified microcrystalline cellulose decorated with coumarin moieties was prepared to design a biobased anti-counterfeiting ink for authentication of original artificial industries based on Förster resonance energy transfer (FRET). For this purpose, the first generation of the PAMAM dendrimer was divergently grown onto the microcrystalline cellulose (MCC) surface and subsequently decorated with coumarin moieties using a Michael addition reaction to obtain a fluorescent hybrid dendrimer. Physical incorporation of fluorescein into the aqueous hybrid dendrimer dispersion resulted in a chemosensor and also an anti-counterfeiting ink based on the FRET mechanism, where the coumarin-decorated hybrid dendrimer acts as a donor and fluorescein plays the role of an acceptor. This aqueous FRET probe based on bioresources was applicable for ion and pH sensing due to the presence of nitrogen atoms on the dendritic structure and also for preparation of anti-counterfeiting inks for use on cellulosic papers, security documents, and, most importantly, artificial industries. This work could help to reduce forgery in artificial industries, authentication of the original handicrafts from the fakes, and prevent from counterfeiting in the art market.

Multiplexed Molecular Tension Sensor Measurements Using PIE-FLIM.

Genetically encoded Förster Resonance Energy Transfer (FRET)-based tension sensors were developed to enable the quantification of piconewton (pN)-scale forces that act across distinct proteins in living cells and organisms. An important extension of this technology is the multiplexing of tension sensors to monitor several independent FRET probes in parallel. Here we describe how pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) can be implemented to enable the analysis of two co-expressed tension sensor constructs. Our protocol covers all essential steps from biosensor expression and live cell PIE image acquisition to lifetime calculations.

Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for theAnalysis of Deubiquitinase Activities.

The genetic code expansion technology enables the genetic encoding of fluorescent noncanonical amino acids (ncAAs) for site-specific fluorescent labeling of proteins. These co-translational and internal fluorescent tags have been harnessed to establish genetically encoded Förster resonance energy transfer (FRET) probes for studying protein structural changes and interactions. Here, we describe the protocols for site-specific incorporation of an aminocoumarin-derived fluorescent ncAA into proteins in E. coli and preparation of a fluorescent ncAA-based FRET probe for assaying theactivities of deubiquitinases, a key class of enzymes involved in ubiquitination. We also describe the deployment of an in vitro fluorescence assay to screen and analyze small-molecule inhibitors against deubiquitinases.

Highly Selective FRET-Aided Single-Molecule Counting of MicroRNAs Labeled by Splinted Ligation.

MicroRNAs (miRNAs) are short non-coding RNAs that play an important role in regulating gene expression. Since miRNAs are abnormally expressed in various cancers, they are considered to be promising biomarkers for early cancer diagnosis. However, the short length and strong sequence similarity among miRNAs make their reliable quantification very challenging. We developed a highly selective amplification-free miRNA detection method based on Förster resonance energy transfer (FRET)-aided single-molecule counting. miRNAs were selectively labeled with FRET probes using splinted ligation. When imaged with a single-molecule FRET setup, the miRNA molecules were accurately identified by the probe's FRET. miRNA concentrations were estimated from the count of molecules. The high sensitivity of the method in finding sparse molecules enabled us to achieve a limit of detection of 31-56 amol for miR-125b, miR-100, and miR-99a. Single nucleotide mismatch could be discriminated with a very high target-to-mismatch ratio. The method accurately measured the high expression of miR-125b in gastric cancer cells, which agreed well with previous reports. The high sensitivity and accuracy of this technique demonstrated its clinical potential as a robust miRNA detection method.

Exploiting the Unique Properties of Lanthanide Complexes as FRET Probes: from Quantitation to Protein Dynamics

AbstractLanthanide complexes have a range of photophysical properties – large pseudo‐Stokes shifts, narrow linewidths, long emission lifetimes – that make them ideal as emissive probes. These same properties also render them excellent donors in assays relying on Förster resonance energy transfer (FRET). Over the last 20 years these assays have increased in sophistication so that a range of biological applications has now been demonstrated. These range from easy‐to‐use quantitation of proteins (a practical alternative to western blotting) to sophisticated experiments which give real‐time information on the movement of proteins or allow simultaneous readout of both agonist and co‐activator binding to a protein. We introduce the principles and difficulties of using such assays before surveying the full scope of what they can achieve and attempting to anticipate future developments in this area.

A Dendrimer-Based Time-Gated Concentric FRET Configuration for Multiplexed Sensing

Förster resonance energy transfer (FRET) is widely used for the development of biological probes and sensors. In this context, the norm for multiplexed detection is deployment of multiple probes, each a discrete donor-acceptor pair. Concentric FRET (cFRET) probes enable multiplexed sensing with a single vector but, to date, have only been developed around semiconductor quantum dots, which may limit the scope of biological applications for such probes. Here, we demonstrate that dendrimers labeled with a luminescent terbium complex (Tb) are a viable and advantageous alternative platform for cFRET probes. Polyamidoamine dendrimers were functionalized with Tb, biotin, NeutrAvidin, and three types of dye-labeled oligonucleotide probes to establish a network of competitive and sequential Tb-to-dye and dye-to-dye FRET pathways. These probes were characterized physically and photophysically, and a time-gated multiplexed assay for DNA targets was demonstrated. The time-gating offered by the Tb allowed the rejection of background autofluorescence from serum. More broadly, this dendrimer-based architecture shows that cFRET is a general concept and is an important step toward a new generation of probes for biological sensing.

Developing FRET Networks for Sensing.

Förster resonance energy transfer (FRET) is a widely used fluorescence-based sensing mechanism. To date, most implementations of FRET sensors have relied on a discrete donor-acceptor pair for detection of each analytical target. FRET networks are an emerging concept in which target recognition perturbs a set of interconnected FRET pathways between multiple emitters. Here, we review the energy transfer topologies and scaffold materials for FRET networks, propose a general nomenclature, and qualitatively summarize the dynamics of the competitive, sequential, homoFRET, and heteroFRET pathways that constitute FRET networks. Implementations of FRET networks for sensing are also described, including concentric FRET probes, other single-vector multiplexing, and logic gates and switches. Unresolved questions and future research directions for current systems are discussed, as are potential but currently unexplored applications of FRET networks in sensing.

Hyperspectral imaging and adaptive thresholding to identify agonist-induced cAMP signals in pulmonary microvascular endothelial cells.

Cyclic AMP (cAMP) is a second messenger that regulates a wide variety of cellular functions. There is increasing evidence suggesting that signaling specificity is due in part to cAMP compartmentalization. In the last 15 years, development of cAMP-specific Förster resonance energy transfer (FRET) probes have allowed us to visualize spatial distributions of intracellular cAMP signals. The use of FRET-based sensors is not without its limitations, as FRET probes display low signal to noise ratio (SNR). Hyperspectral imaging and analysis approaches have, in part, allowed us to overcome these limitations by improving the SNR of FRET measurements. Here we demonstrate that the combination of hyperspectral imaging approaches, linear unmixing, and adaptive thresholding allow us to visualize regions of elevated cAMP (regions of interest - ROIs) in an unbiased manner. We transfected cDNA encoding the H188 FRET-based cAMP probe into pulmonary microvascular endothelial cells. Application of isoproterenol and prostaglandin E1 (PGE1) triggered complex cAMP responses. Spatial and temporal aspects of cAMP responses were quantified using an adaptive thresholding approach and compared between agonist treatment groups. Our data indicate that both the origination sites and spatial/temporal distributions of cAMP signals are agonist dependent in PMVECs. We are currently analyzing the data in order to better quantify the distribution of cAMP signals triggered by different agonists.

Doubling Förster Resonance Energy Transfer Efficiency in Proteins with Extrinsic Thioamide Probes: Implications for Thiomodified Nucleobases.

Designing a potential protein-ligand pair is pivotal, not only to track the protein structure dynamics, but also to assist in an atomistic understanding of drug delivery. Herein, the potential of a small model thioamide probe being used to study albumin proteins is reported. By monitoring the Förster resonance energy transfer (FRET) dynamics with the help of fluorescence spectroscopic techniques, a twofold enhancement in the FRET efficiency of 2-thiopyridone (2TPY), relative to that of its amide analogue, is observed. Molecular dynamics simulations depict the relative position of the free energy minimum to be quite stable in the case of 2TPY through noncovalent interactions with sulfur, which help to enhance the FRET efficiency. Finally, its application is shown by pairing thiouracils with protein. It is found that the site-selective sulfur atom substitution approach and noncovalent interactions with sulfur can substantially enhance the FRET efficiency, which could be a potential avenue to explore in the design of FRET probes to study the structure and dynamics of biomolecules.

Redox-Sensitive Cysteines Confer Proximal Control of the Molecular Crowding Barrier in the Nuclear Pore

The nuclear pore complex forms a highly crowded selective barrier with intrinsically disordered regions at the nuclear membrane to coordinate nucleocytoplasmic molecular communications. Although oxidative stress is known to alter the barrier function, the molecular mechanism underlying this adaptive control of the nuclear pore complex remains unknown. Here we uncover a systematic control of the crowding barrier within the nuclear pore in response to various redox environments. Direct measurements of the crowding states using a crowding-sensitive FRET (Förster resonance energy transfer) probe reveal specific roles of the nuclear pore subunits that adjust the degree of crowding in response to different redox conditions, by adaptively forming or disrupting redox-sensitive disulfide bonds. Relationships between crowding control and the barrier function of the nuclear pore are investigated by single-molecular fluorescence measurements of nuclear transport. Based on these findings, we propose a proximal control model of molecular crowding invivo that is dynamically regulated at the molecular level.

An all-graphene quantum dot Förster resonance energy transfer (FRET) probe for ratiometric detection of HE4 ovarian cancer biomarker

Ovarian cancer (OVC), the most lethal form of all gynecological cancers, is a big threat to women’s health. Late diagnosis at the advanced stages is one of the major reasons for the ovarian cancer-related deaths. Conventionally, the up-regulated proteins CA125 (cancer antigen 125) and HE4 (human epididymis protein 4) are used as biomarkers to diagnose the OVC malignancies. The lack of sensitivity/specificity and the false-positive results create complexity in the diagnostic process. With specificity over 90 %, HE4 is suitable for diagnosing ovarian cancer. Herein, we have developed an ultrasensitive all-graphene quantum dot (GQD) Förster resonance energy transfer (FRET) probe for the ratiometric detection of HE4 biomarker. A set of two GQD samples were solvothermally prepared and then analyzed by the morphological, structural, and photophysical characterization. One GQD sample exhibited a strong green emission, peaked at around 515 nm, while the other GQD sample displayed a strong red emission with maximum at around 615 nm. The good spectral overlap between the emission and excitation spectra of the green and red GQDs, respectively, all allowed us to consider them for the design of FRET-based probe. The green and red-emitting GQDs were conjugated with HE4 antibody and used as donor and acceptor, respectively for the ratiometric sensing of HE4 ovarian cancer biomarker. The all GQD FRET probe was able to detect as low as 4.8 pM, along with a large dynamic detection range up to 300 nM. The selectivity and interference effect of the developed FRET probe was also investigated against different protein combinations.

Visualization of PS/γ-Secretase Activity in Living Cells.

SummaryA change in Presenilin (PS)/γ-secretase activity is linked to essential biological events as well as to the progression of many diseases. However, not much is known about how PS/γ-secretase activity is spatiotemporally regulated in cells. One of the limitations is lack of tools to directly monitor dynamic behavior of the PS/γ-secretase in intact/live cells. Here we present successful development and validation of the Förster resonance energy transfer (FRET)-based biosensors that enable quantitative monitoring of endogenous PS/γ-secretase activity in live cells longitudinally on a cell-by-cell basis. Using these FRET biosensors, we uncovered that PS/γ-secretase activity is heterogeneously regulated among live neurons.