FRET Technique Research Articles

Design, synthesis, in silico and in vitro screening of 1,2,4-thiadiazole analogues as non-peptide inhibitors of beta-secretase

Beta-secretase is the key enzyme involved in Alzheimer’s disease thus; inhibition of the enzyme can lead to a potential anti-Alzheimer drug. In the search of an effective lead candidate, we have designed non-peptide inhibitor molecules based on amino aromatic heterocyclic motifs specifically, substituted 1,2,4-thiadiazole analogues. In silico modelling was employed to study interaction of the designed ligands in the enzyme active site using molecular docking approach as well as for Absorption, Distribution, Metabolism and Excretion studies. The synthesized analogues were pharmacologically screened using in vitro FRET technique. Overall results indicate that one of the analogues, compound 8 is the most promising one against beta secretase.

Brain-SM/SDPC/Cholesterol Phase Diagram

We report the solved 3-component phase diagram for the biologically relevant mixture of brain-SM/ SDPC (18:0,22:6)-PC/cholesterol. Polyunsaturated acyl chains can have special influences on the mixing and phase behaviors of lipid mixtures. Their high degree of unsaturation affects the physical properties of biomembranes in ways that are still not fully understood, and their high concentration in some membranes makes them key players in membrane structure. FRET techniques and fluorescence microscopy imaging of giant unilamellar vesicles (GUVs) were employed for phase boundary visualization and phase identification of this 3-component mixture. Fluorescent lipid probes having complementary partitioning behavior were used to distinguish between phases. Of particular interest is the “raft” region of Ld + Lo phase coexistence, which shows phase separation by FRET methods, yet GUVs appear uniform under microscopy. Taken together these results indicate nanoscopic Ld + Lo phase separation in brain-SM/ SDPC (18:0,22:6)-PC/cholesterol mixture.

Experimental and Computational Studies on the Dynamics and Flexibility of Protein Disulfide-Isomerase (PDI)

PDI is an abundant folding catalyst and chaperone, required for formation of native disulfide bonds in secretory proteins. PDI interacts with many redox partners in the oxidative protein folding pathway and acts on an immense range of unfolded and part-folded protein substrates. It is a multi-domain protein (a-b-b'-x-a'-c) comprising two redox-active thioredoxin-like domains (a, a'), two other domains structurally homologous to thioredoxin (b, b'), a linker (x) and an inessential C-terminal tail (c). Key sites are the redox-active motifs (-WCGHCK-) in a and a' domains, and a promiscuous non-covalent ligand-binding site in b' which can be gated by x. X-ray structures of PDI and its homologues provide static information, but evidence from NMR and intrinsic fluorescence suggests PDI is highly dynamic.We have explored PDI flexibility using FRET and computational techniques. Using yeast PDI, a very rapid method for predicting protein dynamics, based on FRODA and normal mode analysis, has been validated by a comparison of predictions with those from a full MD analysis. Both methods highlight inter-domain motion and show that the character of inter-domain motion at each domain boundary is different, with the x linker providing great freedom for twisting and tilting at the b'-a' interface. Two crystal structures show distinctive twist motion at the a-b interface which is predicted by the rapid method, but not by MD. Similar analyses are now underway on members of the human PDI family.Both theoretical analyses predict that PDI flexibility permits very significant change in the distances between functional sites. We are analysing this experimentally by single-molecule FRET techniques, with FRET probes attached to the redox-active sites and elsewhere. The data indicate that human PDI in solution can access a variety of conformations.

Tracking Voltage-Dependent Conformational Changes of the VSD in Nav with LRET

Voltage-gated sodium channels (Nav) are fundamental for the generation and the propagation of action potentials. Mammalian Nav alpha subunits are single macromolecules organized in four different domains (DI-DIV). Each is composed of 6 transmembrane segments (S1-S6) from which S1-S4 constitute the voltage sensing domain (VSD) and with S5 and S6 constituting the pore. While Nav function has been studied extensively, the exact structural mechanisms of gating are not fully understood. Recently, the crystal structure of the prokaryotic sodium channel, NavAb, has been solved, but NavAb is a homotetrameric protein in contract to the mammalian Navs. Thus many questions were not answered by the prokaryotic channel structures. To resolve the voltage dependent conformational changes of Nav, we tracked conformational changes of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4) using Lanthanide-based Resonance Energy Transfer (LRET), a FRET technique that allows for precise measurement of intermolecular distances by taking advantage of the special properties of lanthanide as an energy donor. We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which holds a lanthanide (Tb3+) ion with high affinity, inserted at the top of the S4 segment in each domain. Also, we synthesized two toxins conjugated to dyes to function as acceptors: the pore-blocking small molecule tetrodotoxin conjugated with a HiLyte fluor488 (TTX-F), and the peptide β scorpion toxin Ts1, from the Brazilian scorpion Tityus serrulatus, conjugated with Alexa488 (Ts1-Alexa488). Having several donor positions (Tb3+ ions in LBT's) and two different acceptor positions (TTX-F and Ts1-Alexa488), we calculated multiple distances in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs that remained functionally active. The results provide new insight to structure-function information in mammalian Nav channels. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

FGF1 and FGF2 Induced FGFR3 Dimerization in Plasma Membrane Derived Vesicles

Fibroblast growth factor receptor 3 (FGFR3) is a receptor tyrosine kinase which resides in the plasma membrane and regulates cell survival, differentiation and angiogenesis. Fibroblast growth factors (fgfs) are a family of secreted protein ligands which bind to FGFRs to potentiate signaling. FGFR3 functions via lateral dimerization in the cellular plasma membrane. It has been shown that FGFR3 forms dimers in the absence of fgf ligands, however it is not clear how fgfs affect FGFR3 dimerization. Previously we have developed a quantitative imaging FRET (QI-FRET) technique to study membrane protein interactions in a cell-derived model system. Here we have used this method to measure thermodynamics of FGFR3 interactions in the presence of fgf1 and fgf2 in Chinese Hamster Ovary (CHO) cell derived vesicles. These measurements provide novel mechanistic insights into the role of ligand binding in receptor tyrosine kinase interactions.

Partial Activation of the Cardiac Myofilament by Ca2+

The cardiac myofilament is a protein assembly that enable the heart to undergo alternating periods of contraction and relaxation, the driving force regulated by Ca2+. Troponin, a three-member protein assembly within the myofilament, acts as a Ca-sensitive switch. In this work, we used single molecule FRET technique to monitor whether the Troponin complex functions as a Ca2+ -sensitive regulatory switch . The results show a population of unactivated troponin under saturating Ca2+ conditions. We propose that the population of unactivated troponin comprises a form of cardiac reserve that is regulated by signaling pathways that target the myofilament.

Structural Dynamics Underlying Pip2 Modulation of Kir Channels Revealed by Single Molecule FRET

Ion channels are gated by a multitude of electrical, chemical and mechanical stimuli, and thereby play key roles in many physiological processes. Static crystal structures and dynamic electrophysiological studies have generated invaluable molecular insights into channel gating, but the time trajectory of conformational changes has been unattainable. KirBac1.1 is a model for one of the three main K channel families. In the present study, we have successfully (1) labeled purified tetrameric protein molecules with single FRET donor and acceptor fluorophore pairs at specific locations; (2) functionally reconstituted the proteins into liposomes; (3) examined the dynamics of channel structure in liposomes with single molecule FRET techniques. Our results indicate that the amphipathic 'slide helices' of KirBac1.1 that surround the channel gate immediately below the membrane, move toward each other to narrow the pore in the presence of PIP2, which closes the channel; the cytoplasmic domain resides at two major structural states, but demonstrates an overall shift away from the pore axis upon PIP2 inhibition; the extracellular loop of KirBac1.1, adjacent to the selectivity filter, is structurally rigid and does not move during PIP2 gating. Both slide helix and cytoplasmic domain structures become less dynamic when the channel is inhibited by PIP2, which implies that PIP2 acts as a 'lock' to confine the structural fluctuations of the channel protein. We further probed the relative motions of the KirBac1.1 transmembrane domain versus the cytoplasmic domain. The results indicate that the entire cytoplasmic domain moves away from the transmembrane domain during PIP2 inhibition, which may result from a rigid body motion coupled to TM2 bending. In summary, we provide direct observations of dynamic structures of an ion channel within a lipid membrane environment, revealing dynamic structural rearrangements KirBac1.1 upon PIP2 inhibition.

Bending of Short dsDNA Upon Binding of Anabaena Sensory Rhodopsin Transducer

Single-molecule fluorescence resonance energy transfer (smFRET) has been used to monitor the intra-molecular changes of DNA or proteins. Practically, the structure information of the macromolecule is required to precisely label the molecules with two fluorescence dyes. The structure information is, however, often not available, which limits the FRET technique from application. In this study, we applied a recently developed single-molecule cyclization assay, to monitor the structural change of double stranded DNA upon binding of Anabaena sensory rhodopsin transducer (ASRT), a novel signal transducer protein involved in photo-sensory and light harvesting system in bacteria whose DNA-bound structure is unknown. We first labeled the two ends of a linear double stranded DNA with a FRET pair, from which the distance between the two ends can be estimated after binding of ASRT. However, FRET would occur only if the two ends are in the FRET range (typically a few nanometers), which cannot be guaranteed without given the structure. To overcome this problem, we placed complementary single stranded tails at the two ends of the DNA, so that the tails may interact and bind to each other and bring the two ends in close proximity. We observed the enhanced loop formation of DNA (∼100 bp) upon binding of ASRT, which suggests that ASRT regulates gene expression by facilitating loop formation of the dsDNA around itself.

Weak Interactions between Risc and mRNA Promote Optimal Targeting in RNA Interference

RNA interference (RNAi) is an essential regulatory process in embryonic development, tumor genesis and other important functions in eukaryotic cells. RNAi occurs when the Argonaute (Ago) protein brings a small non-coding RNA, such as siRNA (small interfering RNA), to target mRNA. Despite a decade of research, it remains an open question how Ago mediates specific targeting inside a crowded cell while it does not utilize any external energy such as ATP.Here we used a single-molecule FRET technique to visualize the target search process of RISC (RNA-induced silencing complex), which consists of human Ago2 and siRNA. We observed that RISC dynamically binds to and dissociates from mRNA. This dynamic interaction occurs even when there is substantial basepairing between siRNA and mRNA. RISC that dissociates from its first target is able to diffuse laterally along the RNA strand and pair with another target nearby.This dynamic search process will lead to an optimal match among many potential pairs. It hints that RISC may have evolved to compromise the search time for the target recognition fidelity. The outcome of our research will be useful in eliminating off-targeting in RNA interference.View Large Image | View Hi-Res Image | Download PowerPoint Slide

New FRET Methods for Studying Processing of Nucleic Acids by Protein Machines

Single-molecule FRET microscopy is a versatile fluorescence technique that serves as a molecular ruler reporting on nanoscale distances and distances changes within biomolecules. During the past 15 years, studies based on single-molecule FRET have uncovered substantial information about the dynamics of nucleic acids (DNA and RNA), as well as the mechanisms of nucleic acid processing by a large variety of protein machines. Such studies are often based on direct, real-time observation of the dynamics of surface-immobilized individual molecules of nucleic acids or their complexes with proteins, and offer first views of intermediates undetectable by more conventional structural and biochemical methods.However, many single-molecule FRET techniques are often limited to a single observable, namely the FRET efficiency between the donor and acceptor probes; as a result, one may need considerable information about a system to interpret the FRET signal appropriately. In my talk, I will describe how the coupling of the FRET signal with additional fluorescence observables at the single-molecule level can be used to explore transient reaction intermediates and reaction paths. Specifically, I will discuss combinations of single-molecule FRET with alternating-laser excitation (ALEX), tethered fluorophore motion (TFM) and protein-induced fluorescence enhancement (PIFE). I will also describe examples of applications of such FRET-based techniques in mechanistic studies of gene transcription and site-specific DNA recombination. Prospects of extending FRET and its combinations in vivo will also be discussed.

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.

Tau-Hsp70 interaction on the single molecule level

Background As the protein quality control centre of the cell molecular chaperones tightly regulate amyloid proteins and seem to alleviate their cytotoxicity. However, the precise mechanism of this interplay and how its modification leads to disease remain elusive. Particularly oligomeric species of amyloid proteins have emerged as the most important mediators of neurotoxicity in neurodegenerative diseases. However, these oligomers are exceedingly hard to characterize as they are transient, heterogeneous and rare relative to the monomeric and fibrillar states. We have developed single molecule FRET techniques for the time-resolved detection and characterisation of oligomeric species [1-3]. Here, we characterise the oligomers evolving during the aggregation of a tau variant carrying the Δ280K mutation associated with some forms of frontotemporal dementia. Further, we study the interaction of these oligomers with the intracellular chaperone Hsp70 which has previously been identified as a key regulator of tau turnover.

Functional Assessment of Crystallization-Optimized G Protein-Coupled Receptors using Ion Channel-Coupled Receptors

Ion Channel-Coupled Receptors (ICCRs) are artificial ligand-gated ion channels created by genetic fusion of G protein-coupled receptors (GPCRs) to a K+ inward rectifier channel (Kir6.2) such that the channel is a direct reporter of the receptor conformational changes. This concept has been validated with 4 prototypical GPCRs: the M2 muscarinic, the D2L dopaminergic, the β2 adrenergic and the opsin receptors (Moreau et al., Nature Nanotech 3:620 2008, Caro et al. PLoS ONE 6:e18226 2011, Caro et al. PLoS ONE 7:e43766 2012). Voltage-clamp recordings showed that ICCRs detect the agonist- and antagonist-bound states of the receptor via direct physical coupling. This GPCR-channel communication proceeds without any involvement of G proteins and the electrical signal amplitude is correlated with the ligand concentration.The intrinsic instability of the GPCRs has proved a challenge to crystallographic studies. A successful approach, introduced in 2007 by Cherezov et al (Science. 318:1258) and subsequently applied to obtain 12 GPCR structures, consists in the insertion of the T4 phage lysozyme domain in the 3rd intracellular loop of the receptors. However, this modification abolishes G protein binding and prohibits related functional assays. Current characterization of crystallization-optimized GPCR(T4L) is performed by radiolabeled ligand assays or localized FRET techniques. Requiring no biochemical steps, ICCRs are an alternative tool to functionally characterize modified GPCRs that are unable to bind or activate G proteins and not amenable to most GPCR functional assays. We demonstrate here the validity of this tool with 3 different GPCRs (M2-muscarinic, β2-adrenergic and oxytocin receptors). The final application of this study would be the integration of this technology in the current crystallographic platforms dedicated to GPCR structure determination or for structure-function studies independent of G protein interaction.

The Existence of FGFR1–5-HT1A Receptor Heterocomplexes in Midbrain 5-HT Neurons of the Rat: Relevance for Neuroplasticity

The ascending midbrain 5-HT neurons to the forebrain may be dysregulated in depression and have a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by coimmunoprecipitation and colocation of the FGFR1 and 5-HT1A immunoreactivities in the midbrain raphe cells, evidence for the existence of FGFR1-5-HT1A receptor heterocomplexes in the dorsal and median raphe nuclei of the Sprague Dawley rat as well as in the rat medullary raphe RN33B cells has been obtained. Especially after combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA clusters was found in the RN33B cells. Similar results were reached with the FRET technique in HEK293T cells, where TM-V of the 5HT1A receptor was found to be part of the receptor interface. The combined treatment with FGF-2 and the 5-HT1A agonist also synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and the RN33B cells as well as their differentiation, as seen from development of the increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TM-V but not by TM-II. Together, the results indicate that the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A receptor heterocomplex in the midbrain raphe 5-HT nerve cells appear to have a trophic role in the central 5-HT neuron systems in addition to playing a key role in reducing the firing of these neurons.

PRION PROTEIN AND AMYLOID BETA PEPTIDE INTERACT IN THE YEAST SACCHAROMYCES CEREVISIAE

SUMMARY: The possibility of interaction between Prion Protein and amyloid beta peptide in living cells of yeast Saccharomyces cerevisiae have been investigated by fluorescence 3D microscopy. Using the FR ET technique, it was shown that amyloid beta peptide and PrP interact in yeast cells. In the future, the yeast model can be used for investigation of the fine mechanisms of this interaction by fluorescence microscopy.

DNA biosensors that reason

Despite the many designs of devices operating with the DNA strand displacement, surprisingly none is explicitly devoted to the implementation of logical deductions. The present article introduces a new model of biosensor device that uses nucleic acid strands to encode simple rules such as “IF DNA_strand1 is present THEN diseaseA” or “IF DNA_strand1 AND DNA_strand2 are present THEN diseaseB”. Taking advantage of the strand displacement operation, our model makes these simple rules interact with input signals (either DNA or any type of RNA) to generate an output signal (in the form of nucleotide strands). This output signal represents a diagnosis, which either can be measured using FRET techniques, cascaded as the input of another logical deduction with different rules, or even be a drug that is administered in response to a set of symptoms. The encoding introduces an implicit error cancellation mechanism, which increases the system scalability enabling longer inference cascades with a bounded and controllable signal–noise relation. It also allows the same rule to be used in forward inference or backward inference, providing the option of validly outputting negated propositions (e.g. “diagnosis A excluded”). The models presented in this paper can be used to implement smart logical DNA devices that perform genetic diagnosis in vitro.

Direct in Vitro and in Vivo Evidence for Interaction between Hsp47 Protein and Collagen Triple Helix

Hsp47 (heat shock protein 47), a collagen-specific molecular chaperone, is essential for the maturation of various types of procollagens. Previous studies have suggested that Hsp47 may preferentially recognize the triple-helix form of procollagen rather than unfolded procollagen chains in the endoplasmic reticulum. However, the underlying mechanism has remained unclear because of limitations in the available methods for detecting in vitro and in vivo interactions between Hsp47 and collagen. In this study, we established novel methods for this purpose by adopting a time-resolved FRET technique in vitro and a bimolecular fluorescence complementation technique in vivo. Using these methods, we provide direct evidence that Hsp47 binds to collagen triple helices but not to the monomer form in vitro. We also demonstrate that Hsp47 binds a collagen model peptide in the trimer conformation in vivo. Hsp47 did not bind collagen peptides that had been modified to block their ability to form triple helices in vivo. These results conclusively indicate that Hsp47 recognizes the triple-helix form of procollagen in vitro and in vivo.

Direct observation of the formation of DNA triplexes by single-molecule FRET measurements

In this report we investigated the effects of various biological and chemical factors (DNA sequence, pH, ions, and molecularity) on the formation of DNA triplexes through single-molecule FRET technique. Using this method, we determined how the third strand bound to a DNA duplex and how stable the triplex structure was under various conditions. From this study, we not only verified a variety of well-known features of DNA triplex but also discovered or experimentally supported several interesting behaviors: at neutral pH, a pyrimidine-motif triplex can be formed; the parallel arrangement was not only possible but also dominant over the antiparallel arrangement for a purine-motif triplex. We demonstrated that our method is a versatile analytical tool in studying structural aspects of nucleic acids, particularly non-classical DNA structures, and provides insights into physical mechanism of such structures.

A simple FRET-based modular design for diagnostic probes

In recent years, there has been a massive effort to develop molecular probes with optical modes of action. Probes generally produce detectable signals based on changes in fluorescence properties. Here, we demonstrate the potential of self-immolative molecular adaptors as a platform for Turn-On probes based on the FRET technique. The probe is equipped with identical fluorophore pairs or a fluorophore/quencher FRET pair and a triggering substrate. Upon reaction of the analyte of interest with the triggering substrate, the self-immolative adaptor spontaneously releases the two dye molecules to break off the FRET effect. As a result, a new measurable fluorescent signal is generated. The fluorescence obtained can be used to quantify the analyte. The modular structure of the probe design will allow the preparation of various chemical probes based on the FRET activation technique.

Single-Molecule Study of RecG Activity at a 3-way DNA Junction

In E. coli, replicative DNA polymerases are often stalled for various reasons: head-on collision of DNA polymerases, blockage by frozen DNA-protein complexes or DNA damage by UV and oxidative stress. In particular, DNA damages causing polymerase stalling should be properly repaired by DNA repair pathways such as NER and HR. In order to avoid the impasse due to polymerase stalling and repair the DNA damages eventually, the stalled fork (3-way junction) is regressed for a proper repair on the damaged site. In the process of fork regression, RecG is thought to play a pivotal role: at the back of the fork, it may pump DNA back in order to convert the 3-way junction to a 4-way chicken-foot structure whereby the damaged spot becomes rewound and prepared for repair process.In our study, we examined the activity of RecG at a 3-way junction at the single-molecule level. Using single-molecule FRET technique, we clarified how RecG unwinds newly-synthesized DNA strands and induces the conformational transition of branched DNA structures. We also investigated the effects of monovalent cation, ATP, and ATP analogs on RecG activity and measured the rate of unwinding of DNA by RecG.From our study, we found in detail how RecG functions: it binds to a 3-way junction resembling a stalled replication fork and unwinds the junction to a 4-way junction in an ATP-dependent manner.