FRET Technique Research Articles

The oligonucleotide-based fluorescence assay for the discovery of tyrosyl-DNA phosphodiesterase 2 inhibitors

Tyrosyl-DNA phosphodiesterase 2 (TDP2) plays an important role in the repair of DNA damage induced by Topoisomerase II (TOP2), and is a potential anticancer target. In this work, we established an efficient and precise fluorescence assay for TDP2 inhibitor discovery based on FRET technique. The sequent screening of our natural product library led to the identification of a new TDP2 inhibitor 3 (8.6 ± 0.35 μM), which showed synergistic effect with etoposide in HCT116 and DU145 cell lines. The possible binding mode of 3 with TDP2 protein was also studied.

Mechanistic insights into the 2-step dimerization process FO the hepatitis C virus RNA genome.

The 3ʹX RNA of the Hepatitis C Virus (HCV) is a highly conserved, 98-nt sequence located at the 3ʹ terminus of the HCV genome. This region of the genome can engage in several viral RNA-RNA binding interactions that have previously been shown to be critical for regulating translation, replication, and viral particle assembly. Within the 3ʹX RNA there is a stretch of 16 nucleotides that is responsible for two different RNA-RNA binding interactions: one heterotypic and the other homotypic. Notably, the nucleotide sequence for the heterotypic interaction is fully nested within the homotypic interaction and therefore formation of one interaction precludes formation of the other. Although these two interactions have important regulatory roles, it is not known what determines which interaction is formed at any given time and how they might interconvert. Using single-molecule FRET (smFRET) and size exclusion HPLC (SE-HPLC) techniques to monitor the conformational status of the 3ʹX RNA under a variety of conditions, we are beginning to resolve these unanswered questions. First, in the absence of either binding partner, we observe two distinct conformations of monomeric 3ʹX with their relative abundance dictated by either solution conditions (e.g., [Mg2+]) or nucleotide deletions. Additionally, we a observe large conformational change in the 3ʹX RNA when exposed to its homotypic binding partner, resulting in the formation of a homodimeric extended duplex of the 3ʹX RNA. These findings support an emerging hypothesis that the conformation changes associated with the 3ʹX RNA function as riboregulatory switches that halt protein and RNA synthesis, ultimately giving way for virus particles to begin to assemble.

An integrative approach to molecular dynamics and single molecule FRET techniques.

Molecular dynamics (MD) simulation and single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy are two techniques that can be used to study conformational heterogeneity of proteins. However, significant challenges exist in these techniques that limit their effectiveness. All-atom MD is currently too slow to adequately sample conformational space of proteins. From an experimental perspective, deciding where to place the fluorescent dye reporters for smFRET measurements can lead to a great deal of wasted time, effort, and expense in producing labeled proteins that may or may not provide useful information. Even if the dyes are suitably placed, interpretation of smFRET data is also challenging since many conformations can lead to the same FRET efficiency for a single smFRET-labeled protein. In this work, we have addressed these problems using a highly integrative approach to smFRET and MD. We have developed an iterative method to characterize the conformational ensemble of proteins by combining MD-based enhanced sampling techniques and smFRET spectroscopy, where the computational and experimental approaches can benefit mutually from each other. We first employed an enhanced sampling based MD method (ES-MD) using system-specific collective variables for the system under study to sample various conformations. Since these simulations are biased, the relative weight of different populations of samples may not be accurate. In the next step, we predicted the FRET efficiency distributions for a large number of residue pairs and from the imperfect results determined the residues that are predicted to provide the most informative FRET distributions. The measured smFRET data was then used within a resampling scheme to correct the original computed set of conformations, which was then used to predict additional smFRET distributions for other labeled residues. We have examined this methodology on intrinsically-disordered/structured and membrane/soluble proteins.

Deciphering Regulatory Proteins of Prenylated Protein via the FRET Technique Using Nitroso-Based Ene-Ligation and Sequential Azidation and Click Reaction.

We report here the selective incorporations of nitroso species into a wide range of proteins targeting lysine residue(s). The corresponding azo functionalities were formed in a highly selective manner with excellent yields, displaying rather good stability under physiological conditions. Furthermore, the azodation proceeded smoothly in high yields on targeted peptides. Fluorescent and/or dual fluorescent labeling of varied proteins following this protocol have been determined efficiently and selectively. With this established protocol, we aim to determine its usage in the evaluation of the interaction of prenylated proteins with their interacted enzyme(s) via FRET assays. Delightedly, chemically modified proteins with a 1-pyrenyl fluorophore through 254 nm UV irradiation and the sequential azodation and click reaction of protein prenyl functionality, which enable the incorporation of naphthene, indeed increase the fluorescence energy transferred since we observed significantly enhanced absorption located at 218 nm in lysed HEK293T cells and a clearly strengthened greenish fluorescence in living HEK293T cells.

New insight into the mechanism of DNA polymerase I revealed by single-molecule FRET studies of Klenow fragment

We use single-molecule FRET and newly-developed D-loop techniques to investigate strand displacement activity of Klenow fragment (exo-) of DNA polymerase I in DNA sequences rich in guanine and cytosine (GC) bases. We find that there exist in the FRET traces numerous ascending jumps, which are induced by the backsliding of Klenow fragment on DNA chains. Our measurements show that the probability of backsliding is closely related to the GC-richness and dNTP concentration: increasing the GC-richness leads to an increase in the backsliding probability, and increasing the dNTP concentration however leads to a decrease in the backsliding probability. These results provide a new insight into the mechanism of DNA polymerase I.

An In Vivo Fluorescence Resonance Energy Transfer-Based Imaging Platform for Targeted Drug Discovery and Cancer Therapy.

In the present study, an efficient in vivo drug screening platform is established based on FRET technique. We transfected cancer cells with FRET-based caspase-3 (C3) sensor and validated the cell lines by detecting the change in FRET signal caused by the in vitro drug-induced cell apoptosis. Furthermore, the C3 expressing cancer cells were then injected into zebrafish embryos and nude mice to establish the corresponding in vivo xenograft models. We found that cancer cell lines expressing C3 were effective in detecting cell death following drug treatment, including the detection of the tipping point of apoptosis. The drug-induced cell apoptosis was also observed in both zebrafish embryos and nude mice xenograft models. Overall, the FRET-based platform, through in vivo imaging, is potentially useful to improve drug screening efficiency.

Spatial and temporal dynamics of RhoA activities of single breast tumor cells in a 3D environment revealed by a machine learning-assisted FRET technique

One of the hallmarks of cancer cells is their exceptional ability to migrate within the extracellular matrix (ECM) for gaining access to the circulatory system, a critical step of cancer metastasis. RhoA, a small GTPase, is known to be a key molecular switch that toggles between actomyosin contractility and lamellipodial protrusion during cell migration. Current understanding of RhoA activity in cell migration has been largely derived from studies of cells plated on a two-dimensional (2D) substrate using a FRET biosensor. There has been increasing evidence that cells behave differently in a more physiologically relevant three-dimensional (3D) environment. However, studies of RhoA activities in 3D have been hindered by low signal-to-noise ratio in fluorescence imaging. In this paper, we present a a machine learning-assisted FRET technique to follow the spatiotemporal dynamics of RhoA activities of single breast tumor cells (MDA-MB-231) migrating in a 3D as well as a 2D environment. We found that RhoA activity is more polarized along the long axis of the cell for single cells migrating on 2D fibronectin-coated glass versus those embedded in 3D collagen matrices. In particular, RhoA activities of cells in 2D exhibit a distinct front-to-back and back-to-front movement during migration in contrast to those in 3D. Finally, regardless of dimensionality, RhoA polarization is found to be moderately correlated with cell shape.

Assessing chromatin condensation for epigenetics with a DNA-targeting sensor by FRET and FLIM techniques

Here we propose a fluorescent sensor, Chroma-V, consisted of a Hoechst ligand (Hoe) to target chromatin DNA and a BODIPY rotor (BDP) to sense the local viscosity that reflects chromatin condensation state. Within Chroma-V, efficient FRET process from Hoe to BDP facilitated a single-excitation ratiometric imaging of nucleus DNA under fluorescence confocal microscope, which utilized the ratio of two channels to enable an intuitive visualization of chromatin condensation state. And fluorescence lifetime imaging (FLIM) based on fluorescent signal from BDP proved to be a more accurate method to quantify the changes of chromatin condensation state under different epigenetic states, including histone acetylation regulated by deacetylase inhibitors, cell apoptosis induced by DNA-bining drugs, and the epithelial-mesenchymal transition of HUVEC cells induced by TGF-β.

Insight into Sodium Pump Regulation by FXYD Proteins

The sodium pump (Na+/K+-ATPase, NKA) is a membrane transport ATPase responsible for establishing the vital gradients of sodium and potassium across the plasma membrane of all mammalian cells. NKA activity is regulated by FXYD proteins, a family of transmembrane proteins that serve as auxiliary subunits of the sodium pump. in the heart, NKA activity is modulated by phospholemman (FXYD1), but the structural determinants of this regulatory mechanism are incompletely understood. We used time-resolved and steady-state FRET techniques to study the stoichiometry and affinity of fluorescently labeled NKA and FXYD1. Progressive acceptor photobleaching revealed multiple acceptor-labeled FXYD1 bound to donor-labeled NKA. Complementary global analysis of time-correlated single photon counting experiments revealed presence of medium- and high-FRET species within the population of NKA-FXYD1 complexes. To explore the quaternary structure of the regulatory complex, we obtained fluorescence lifetime data from hundreds of cells spanning a wide range of protein expression level. Over this range, the high FRET species increased with increasing protein concentration at the expense of the medium-FRET population. We attribute these subpopulations to NKA-NKA dimers decorated with either one FXYD1 (at low concentration) or two (at high concentration). Moreover, acceptor photobleaching experiments revealed FRET between donor- and acceptor-labeled NKA α subunits consistent with NKA oligomerization. in addition, we quantified FRET between NKA α and β essential subunits to monitor changes in the dynamic equilibrium between open (E2) and closed (E1) states of NKA. Treatment of NKA with the specific inhibitor ouabain induced a shift in the dynamic equilibrium of NKA toward a higher FRET state, suggesting that the α and β n-termini moved closer together. The experiments reveal new insight into the regulation of NKA by FXYD proteins and by NKA oligomerization.

The human-specific duplicated α7 gene inhibits the ancestral α7, negatively regulating nicotinic acetylcholine receptor-mediated transmitter release

Gene duplication generates new functions and traits, enabling evolution. Human-specific duplicated genes in particular are primary sources of innovation during our evolution although they have very few known functions. Here we examine the brain function of one of these genes (CHRFAM7A) and its product (dupα7 subunit). This gene results from a partial duplication of the ancestral CHRNA7 gene encoding the α7 subunit that forms the homopentameric α7 nicotinic acetylcholine receptor (α7-nAChR). The functions of α7-nAChR in the brain are well defined, including the modulation of synaptic transmission and plasticity underlying normal attention, cognition, learning, and memory processes. However, the role of the dupα7 subunit remains unexplored at the neuronal level. Here, we characterize that role by combining immunoblotting, quantitative RT-PCR and FRET techniques with functional assays of α7-nAChR activity using human neuroblastoma SH-SY5Y cell variants with different dupα7 expression levels. Our findings reveal a physical interaction between dupα7 and α7 subunits in fluorescent protein-tagged dupα7/α7 transfected cells that negatively affects normal α7-nAChR activity. Specifically, in both single cells and cell populations, the [Ca2+]i signal and the exocytotic response induced by selective stimulation of α7-nAChR were either significantly inhibited by stable dupα7 overexpression or augmented after silencing dupα7 gene expression with specific siRNAs. These findings identify a new role for the dupα7 subunit as a negative regulator of α7-nAChR-mediated control of exocytotic neurotransmitter release. If this effect is excessive, it would result in an impaired synaptic transmission that could underlie the neurocognitive and neuropsychiatric disorders associated with α7-nAChR dysfunction.

Super-resolution FRET measurements

Super-resolution fluorescence microscopy and Förster Resonance Energy Transfer (FRET) form a well-established family of techniques that has provided unique tools to study the dynamic architecture and functionality of biological systems, as well as to investigate nanomaterials. In the last years, the integration of super-resolution methods with FRET measurements has generated advances in two fronts. On the one hand, FRET-based probes have enhanced super-resolution imaging. On the other, the development of super-resolved FRET imaging methods has allowed the visualization of molecular interaction patterns with higher spatial resolution, less averaging and higher dynamic range. Here, we review these advances and discuss future perspectives, including the possible integration of FRET with next generation super-resolution techniques capable of reaching true molecular-scale spatial resolution.

International Council for Standardization in Haematology (ICSH) recommendations for laboratory measurement of ADAMTS13.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH), by the ADAMTS13 Assay Working Group, which comprises an international group of both clinical and laboratory experts. The document provides recommendations on best practice for the performance of ADAMTS13 assays in clinical laboratories. ADAMTS13 assays support the differential diagnosis of thrombotic microangiopathies and have utility in the management of thrombotic thrombocytopenic purpura (TTP). There are three types of assay: activity, antigen and autoantibody/inhibitor assays. Methods for activity assays differ in terms of sensitivity, specificity, precision and turnaround time. The most widely used assays involve VWF peptide substrates and either chromogenic ELISA or FRET techniques, although chemiluminescence assays and rapid screening tests have recently become available. Tests for autoantibodies and inhibitors allow confirmation of acquired, immune-mediated TTP, while antigen assays may be useful in congenital TTP and as prognostic markers. In this document, we have attempted to describe ADAMTS13 assays and the conditions that affect them, as well as: blood collection, sample processing, quality control, standardization and clinical utility; recognizing that laboratories in different parts of the world have varying levels of sophistication. The recommendations are based on expert opinion, published literature and good clinical laboratory practice.

Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2.

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Determining Protein Structures by Iterating between Computation and Experiment

An increasing variety of experimental techniques for protein structure determination rely on the site-specific incorporation of probes. Examples include NMR using the paramagnetic relaxation enhancement (PRE) effect, fluorescence using FRET and related techniques, and site-directed chemical cross-linking quantified by mass-spectrometry. In each case, the probe locations must be carefully chosen, which poses a major challenge, as some probe locations are highly informative, whereas others yield little useful information. In the absence of structural knowledge, how does one efficiently choose probe locations? We present a method called Rational Probe Mutagenesis (RPM) that relies on iteration between computation and experiment to rapidly determine protein structures. RPM is based on the idea of reducing the “width” of the conformational ensemble as rapidly as possible. An ensemble is generated using whatever data is currently available. This ensemble is then analyzed to find the experiment that maximally discriminates between possible structures. The resulting experimental data is then used to generate a new updated ensemble and the process repeated. We demonstrate RPM on several simple model systems and a challenging real-world solid-state NMR dataset, where we show that RPM can choose highly informative experiments, leading to potential time and cost savings by avoiding redundant or uninformative experiments.

DNA replication initiation in Bacillus subtilis: structural and functional characterization of the essential DnaA-DnaD interaction.

The homotetrameric DnaD protein is essential in low G+C content gram positive bacteria and is involved in replication initiation at oriC and re-start of collapsed replication forks. It interacts with the ubiquitously conserved bacterial master replication initiation protein DnaA at the oriC but structural and functional details of this interaction are lacking, thus contributing to our incomplete understanding of the molecular details that underpin replication initiation in bacteria. DnaD comprises N-terminal (DDBH1) and C-terminal (DDBH2) domains, with contradicting bacterial two-hybrid and yeast two-hybrid studies suggesting that either the former or the latter interact with DnaA, respectively. Using Nuclear Magnetic Resonance (NMR) we showed that both DDBH1 and DDBH2 interact with the N-terminal domain I of DnaA and studied the DDBH2 interaction in structural detail. We revealed two families of conformations for the DDBH2-DnaA domain I complex and showed that the DnaA-interaction patch of DnaD is distinct from the DNA-interaction patch, suggesting that DnaD can bind simultaneously DNA and DnaA. Using sensitive single-molecule FRET techniques we revealed that DnaD remodels DnaA–DNA filaments consistent with stretching and/or untwisting. Furthermore, the DNA binding activity of DnaD is redundant for this filament remodelling. This in turn suggests that DnaA and DnaD are working collaboratively in the oriC to locally melt the DNA duplex during replication initiation.

Sarcomere length-dependent effects on Ca2+-troponin regulation in myocardium expressing compliant titin.

Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca2+ concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca2+-induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin-actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca2+-induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca2+-troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length-dependent enhancement of troponin regulation with both Ca2+ activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length-dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length-dependent Ca2+-troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca2+-troponin regulation of the myocardium.

The EphA2 receptor is activated through induction of distinct, ligand-dependent oligomeric structures

The EphA2 receptor tyrosine kinase is capable of activating multiple diverse signaling pathways with roles in processes such as tissue homeostasis and cancer. EphA2 is known to form activated oligomers in the presence of ephrin-A ligands. Here, we characterize the lateral interactions between full-length EphA2 molecules in the plasma membrane in the presence of three types of ligands (dimeric ephrinA1-Fc, monomeric ephrinA1, and an engineered peptide ligand) as well as in the absence of ligand, using a quantitative FRET technique. The data show that EphA2 forms higher-order oligomers and two different types of dimers that all lead to increased EphA2 tyrosine phosphorylation, which is indicative of increased kinase-dependent signaling. We find that different ligands stabilize conformationally distinct oligomers that are assembled through two different interfaces. Our results suggest that these different oligomeric assemblies could have distinct signaling properties, contributing to the diverse activities of the EphA2 receptor.

A Photoswitchable Dualsteric Ligand Controlling Receptor Efficacy.

The investigation of the mode and time course of the activation of G-protein-coupled receptors (GPCRs), in particular muscarinic acetylcholine (mACh or M) receptors, is still in its infancy despite the tremendous therapeutic relevance of M receptors and GPCRs in general. We herein made use of a dualsteric ligand that can concomitantly interact with the orthosteric, that is, the neurotransmitter, binding site and an allosteric one. We synthetically incorporated a photoswitchable (photochromic) azobenzene moiety. We characterized the photophysical properties of this ligand called BQCAAI and investigated its applicability as a pharmacological tool compound with a set of FRET techniques at the M1 receptor. BQCAAI proved to be an unprecedented molecular tool; it is the first photoswitchable dualsteric ligand, and its activity can be regulated by light. We also applied BQCCAI to investigate the time course of several receptor activation processes.

Lipid Bilayer Association of the Alternatively Translated Region of PTEN-Long

PTEN-Long is a 576-amino acid translational variant of phosphatase and tension homolog on chromosome ten (PTEN), a tumor suppressor gene and antagonist to phosphoinositide-3-kinase (PI3K) signaling. PTEN-Long (PTEN-L) exhibits at the N-terminal end an alternative translation region (ATR) with additional 173 N-terminal amino acids to the normal PTEN open reading frame. PTEN-L is secreted from cells, can exist outside the cell and shows activity towards PI(3,4,5)P3 after cell entry. Therefore, PTEN-L may have therapeutic uses by restoring a functional tumor suppressor protein to tumor cells. The mechanism by which PTEN-L enters cells is currently unknown. The ATR region of PTEN-L is evolutionary conserved and contains a polyarginine stretch with homology to cell permeable peptides, suggesting that PTEN-L may be able to traverse the plasma membrane passively. It has been suggested that the N-terminal ATR part of PTEN-L is intrinsically disordered, which we confirmed using CD and FTIR spectroscopy. Our FTIR studies did not reveal any observable structural change of PTEN-L ATR upon lipid binding to PC, PS, and PI(4,5)P2. We further found that the integrity of model lipid bilayers is compromised upon interaction of PTEN-L's ATR. We are currently further delineating the lipid binding preferences of the ATR part of PTEN-L, as well as the full-length protein using surface plasmon resonance (SPR) and FRET techniques.

Sarcomere Lengthdependent Effects on the Ca2+-Troponin Regulation in Skinned Myocardial Fiber from Titin RBM20 Deletion Mice

The length dependent activation of the myofilaments has been considered the cellular basis underlying the Frank-Starling law of the heart trough dynamic and complex interplays between a multitude of thick- and thin-filament components. Among these components, both troponin and giant protein titin are likely key players, but the mechanism, by which the length dependent myofilament Ca2+ regulation is linked functionally to titin, is elusive. In this study we investigate the link using in situ FRET technique to monitor how the length dependent Ca2+-induced conformational change of the N-domain of cTnC within myocardium is modulated by alteration of titin compliance that directly determines the passive tension of myocardium. To achieve the objective, FRET donor and acceptor modified cTnC(13C/51C)AEDANS-DDPM was reconstituted into chemically-skinned myocardial fibers from wild-type mice and RBM20 deficient mice that expresses more compliant N2BA titin. The Ca2+-induced conformational change in cTnC under different conditions were quantitatively characterized using time-resolved FRET measurements. Our results from wild type myocardium showed that sarcomere length dependent enhancement of Ca2+-troponin regulation was found to be directly associated with strong crossbridge and it was diminished when the strong crossbridge was inhibited. When measurements were performed with skinned myocardial fibers from RBM20 deletion animals, the length dependent change was blunted. These results support that titin based passive tension development of myocardium is indeed involved in sarcomere length modulated Ca2+-troponin regulation and that the strong crossbridge plays an important role in linking the modulatory effect of titin compliance to Ca2+-troponin regulation.