Intramolecular Fluorescence Resonance Energy Transfer Research Articles

Developing Chemical Genetic Approaches to Explore G Protein-Coupled Receptor Function: Validation of the Use of a Receptor Activated Solely by Synthetic Ligand (RASSL)

Molecular evolution and chemical genetics have been applied to generate functional pairings of mutated G protein-coupled receptors (GPCRs) and nonendogenous ligands. These mutant receptors, referred to as receptors activated solely by synthetic ligands (RASSLs) or designer receptors exclusively activated by designer drugs (DREADDs), have huge potential to define physiological roles of GPCRs and to validate receptors in animal models as therapeutic targets to treat human disease. However, appreciation of ligand bias and functional selectivity of different ligands at the same receptor suggests that RASSLs may signal differently than wild-type receptors activated by endogenous agonists. We assessed this by generating forms of wild-type human M(3) muscarinic receptor and a RASSL variant that responds selectively to clozapine N-oxide. Although the RASSL receptor had reduced affinity for muscarinic antagonists, including atropine, stimulation with clozapine N-oxide produced effects very similar to those generated by acetylcholine at the wild-type M(3)-receptor. Such effects included the relative movement of the third intracellular loop and C-terminal tail of intramolecular fluorescence resonance energy transfer sensors and the ability of the wild type and evolved mutant to regulate extracellular signal-regulated kinase 1/2 phosphorylation. Each form interacted similarly with β-arrestin 2 and was internalized from the cell surface in response to the appropriate ligand. Furthermore, the pattern of phosphorylation of specific serine residues within the evolved receptor in response to clozapine N-oxide was very similar to that produced by acetylcholine at the wild type. Such results provide confidence that, at least for the M(3) muscarinic receptor, results obtained after transgenic expression of this RASSL are likely to mirror the actions of acetylcholine at the wild type receptor.

Nucleosome Linker DNA Contacts and Induces Specific Folding of the Intrinsically Disordered H1 Carboxyl-Terminal Domain

Linker histones play essential roles in the chromatin structure of higher eukaryotes. While binding to the surface of nucleosomes is directed by an ∼ 80-amino-acid-residue globular domain, the structure and interactions of the lysine-rich ∼ 100-residue C-terminal domain (CTD), primarily responsible for the chromatin-condensing functions of linker histones, are poorly understood. By quantitatively analyzing binding of a set of H1 CTD deletion mutants to nucleosomes containing various lengths of linker DNA, we have identified interactions between distinct regions of the CTD and nucleosome linker DNA at least 21 bp from the edge of the nucleosome core. Importantly, partial CTD truncations caused increases in H1 binding affinity, suggesting that significant entropic costs are incurred upon binding due to CTD folding. van't Hoff entropy/enthalpy analysis and intramolecular fluorescent resonance energy transfer (FRET) studies indicate that the CTD undergoes substantial nucleosome-directed folding, in a manner that is distinct from that which occurs upon H1 binding to naked DNA. In addition to defining critical interactions between the H1 CTD and linker DNA, our data indicate that the H1 CTD is an intrinsically disordered domain and provide important insights into the biological function of this protein.

Cross-talk between Carboxypeptidase M and the Kinin B1 Receptor Mediates a New Mode of G Protein-coupled Receptor Signaling

G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys(9)-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase.

Convenient and Efficient FRET Platform Featuring a Rigid Biphenyl Spacer between Rhodamine and BODIPY: Transformation of ‘Turn‐On’ Sensors into Ratiometric Ones with Dual Emission

We have connected a borondipyrromethene (BODIPY) donor to the 5' position of a tetramethylrhodamine (TMR) acceptor to form a high efficiency (over 99%) intramolecular fluorescence resonance energy transfer (FRET) cassette, BODIPY-rhodamine platform (BRP). While the good spectral overlap between the emission of BODIPY and the absorption of TMR was one favorable factor, another feature of this FRET system was the rigid and short biphenyl spacer that favored efficient through-bond energy transfer. More importantly, in this system, the 2'-carboxyl group of the rhodamine unit was preserved for the further modifications, which was as convenient as those carbonyl groups on the original rhodamines without connection to donors. For this reason, BRP is clearly differentiated from the previous ratiometric sensors based on donor rhodamine systems. To illustrate its value as a versatile platform, we introduced typical Hg(2+) receptors into BRP, through convenient one-pot reactions on the 2'-carboxyl group, and successfully developed two ratiometric sensors, BRP-1 and BRP-2, with different spirocyclic receptors that recognized Hg(2+) on different reaction mechanisms. Upon excitation at a single wavelength (488 nm), at which only BODIPY absorbed, both of the FRET sensors exhibited clear Hg(2+)-induced changes in the intensity ratio of the two strong emission bands of BODIPY and rhodamine. It should be noted that these ratiometric Hg(2+) sensors exhibited excellent sensitivity and selectivity Hg(2+), as well as pH insensitivity, which was similar to the corresponding 'turn-on' rhodamine sensors. While both ratiometric probes were applicable for Hg(2+) imaging in living cells, BRP-1 exhibited higher sensitivity and faster responses than BRP-2. Our investigation indicated that on a versatile platform, such as BRP, a large number of highly efficient ratiometric sensors for transition-metal ions could be conveniently developed.

Conformational Changes of SERCA Revealed by Intramolecular FRET

The first structures of the sarcoendoplasmic reticulum ATPase (SERCA) determined by X-ray crystallography suggested that pump undergoes a large conformational change during catalytic cycling. The transition from the E1 (Ca-bound) state to the E2 (Ca-free) state was predicted to decrease inter-domain separation distance with closure of the SERCA cytoplasmic headpiece. To test this model, we fused Cerulean to the A-domain and YFP to the N or P or TM-domain of SERCA2a. These “2-color” SERCA constructs were expressed in AAV cells, and SERCA structure transitions were detected by changes in intramolecular fluorescence resonance energy transfer (FRET). FRET decreased with thapsigargin for the two N-domain fusion sites (residues 510, 577), while the P- (residue 610) and TM- domain (C-terminus) fusions showed increased FRET with thapsigargin. Unexpectedly, FRET in permeabilized cells was higher in Ca [10 μM] than in EGTA for all constructs, suggesting an increase in domain separation distance with the E1 to E2 transition. These observations were supported by parallel experiments in fluorescence lifetime distribution (FLD) analysis. FLD resolved two broad distributions of fluorescence lifetimes for 2-color SERCA expressed in live cardiac myocytes, consistent with two major FRET states. The relative populations of these states oscillated with electrical pacing, favoring the high FRET (short distance) state during systole (contraction) and the low FRET (long distance) state during diastole (relaxation). We expect 2-color SERCA constructs to be useful for exploring the magnitude, direction, and kinetics of calcium pump conformational changes. They may also be useful for screening candidate compounds for modulation of SERCA pump structure and function.

The Role of Initiation Factor 3 Structural Dynamics in Regulating the Fidelity of Translation Initiation

Initiation factor (IF) 3 is a protein translation factor that, together with IF1 and IF2, controls the selection of a unique initiator tRNA and the correct messenger RNA start codon by the small (30S) ribosomal subunit and ensures that the subsequent association of the large (50S) ribosomal subunit is selectively accelerated only in response to a correctly initiated 30S subunit. IF3 consists of globular N- and C-terminal domains that are separated by a flexible linker, thus it is a structurally dynamic protein and, perhaps owing to these dynamics, decades of genetic, biochemical, and structural studies have thus far failed to provide a widely accepted mechanism through which IF3 regulates initiator tRNA and start codon selection and 50S subunit association. In order to explore the possible role of IF3 dynamics in controlling these processes, we have constructed an IF3 variant carrying fluorescence resonance energy transfer (FRET) donor and acceptor fluorophores within its globular N- and C-terminal domains and have developed an intramolecular IF3 FRET signal. Using single-molecule FRET imaging, we are using this IF3 signal to investigate the intramolecular dynamics of 30S subunit-bound IF3 during translation initiation. Our results demonstrate that 30S subunit-bound IF3 can access multiple conformational sub-states whose occupancies are regulated by the presence of IF1 and/or IF2 on the 30S subunit as well as by the presence and identity of the tRNA and codon located within the 30S subunit peptidyl-tRNA binding site. Most importantly, we find that a fully assembled and correctly initiated 30S subunit uniquely stabilizes a single IF3 conformational sub-state which we hypothesize serves to permit and/or promote 50S subunit association.

Bidirectional Fluorescence Resonance Energy Transfer (FRET) in Mutated and Chemically Modified Yellow Fluorescent Protein (YFP)

Fluorescence resonance energy transfer (FRET) using fluorescent protein variants are used for studying the associations and biomolecular motions of macromolecules inside the cell. Intramolecular FRET utilizing fluorescent chemical labels has been applied in nucleic acid chemistry for detection of specific sequence. However, the biotechnological applications of intramolecular FRET in fluorescent proteins have not been exploited. This study demonstrates the intramolecular FRET between fluorescent protein and conjugated chemical label whereby FRET occurs from inside to outside and vice versa for fluorescent protein. The fluorescent protein is modified for the attachment of chemical fluorophores and the novel FRET pairs created by conjugation are MDCC (435/475)-Citrine (516/529) and Citrine-Alexa fluor (568/603). These protein-label pairs exhibited strong intramolecular FRET and the energy transfer efficiency was determined based on the time evolution of the ratio of emission intensities of labeled and unlabeled proteins. The efficiency was found to be 0.79 and 0.89 for MDCC-Citrine and 0.24 and 0.65 for Citrine-Alexa Fluor pairs when the label is conjugated at different sites in the protein. Förster distance and the average distance between the fluorophores were also determined. The bidirectional approach described here can provide new insights into designing FRET-based sensors.

Simultaneous Intracellular β-d-Glucosidase and Phosphodiesterase I Activities Measurements Based on A Triple-Signaling Fluorescent Probe

Despite that considerable efforts have been devoted to the design of various fluorogenic enzyme substrates, they are single-enzyme assay approaches that cannot afford detection of multienzyme activity. In this study, we set out our first attempt to design a new probe that could measure intracellular β-D-glucosidase and phosphodiesterase I activities. Unlike the commonly used fluorogenic enzyme substrates that contain one recognition site and signaling reporter, the new probe molecule possesses two cleavage sites, specificly corresponding to β-D-glucosidase and phosphodiesterase I, and three fluorescent reporters, 7-β-D-glucopyranosyloxycoumarin, 7-hydroxycoumarin, and meso-tetraphenylporphyrin. On the basis of intramolecular photoinduced electron transfer and fluorescence resonance energy transfer mechanisms, interaction of the probe with the two enzymes, whether only one or both, produces different signal readouts with high sensitivity. Remarkably, the probe is chemically stable in complex biological fluids. Fluorescence outputs are not significantly affected by biologically related metal ions, anions, amino acids, and proteins. Furthermore, fluorescence microscopy confirmed that the probe is an excellent candidate for intracellular delivery and can be accumulated intensively in cells. We demonstrated the applicability for the simultaneous images of intracellular β-D-glucosidase and phosphodiesterase I activities using the different optical imaging modes.

Quantitative analysis of energy transfer between fluorescent proteins in CFP–GBP–YFP and its response to Ca2+

This article reports the full characterisation of the optical properties of a biosynthesised protein consisting of fused cyan fluorescent protein, glucose binding protein and yellow fluorescent protein. The cyan and yellow fluorescent proteins act as donors and acceptors for intramolecular fluorescence resonance energy transfer. Absorption, fluorescence, excitation and fluorescence decays of the compound protein were measured and compared with those of free fluorescent proteins. Signatures of energy transfer were identified in the spectral intensities and fluorescence decays. A model describing the fluorescence properties including energy transfer in terms of rate equations is presented and all relevant parameters are extracted from the measurements. The compound protein changes conformation on binding with calcium ions. This is reflected in a change of energy transfer efficiency between the fluorescent proteins. We track the conformational change and the kinetics of the calcium binding reaction from fluorescence intensity and decay measurements and interpret the results in light of the rate equation model. This visualisation of change in protein conformation has the potential to serve as an analytical tool in the study of protein structure changes in real time, in the development of biosensor proteins and in characterizing protein-drug interactions.

Detection of enzyme activity in orthotopic murine breast cancer by fluorescence lifetime imaging using a fluorescence resonance energy transfer–based molecular probe

Cancer-related enzyme activity can be detected noninvasively using activatable fluorescent molecular probes. In contrast to "always-on" fluorescent molecular probes, activatable probes are relatively nonfluorescent at the time of administration due to intramolecular fluorescence resonance energy transfer (FRET). Enzyme-mediated hydrolysis of peptide linkers results in reduced FRET and increase of fluorescence yield. Separation of signal from active and inactive probe can be difficult with conventional intensity-based fluorescence imaging. Fluorescence lifetime (FLT) measurement is an alternative method to detect changes in FRET. Thus, we investigate FLT imaging for in vivo detection of FRET-based molecular probe activation in an orthotopic breast cancer model. Indeed, the measured FLT of the enzyme-activatable molecular probe increases from 0.62 ns just after injection to 0.78 ns in tumor tissue after 4 h. A significant increase in FLT is not observed for an always-on targeted molecular probe with the same fluorescent reporter. These results show that FLT contrast is a powerful addition to preclinical imaging because it can report molecular activity in vivo due to changes in FRET. Fluorescence lifetime imaging exploits unique characteristics of fluorescent molecular probes that can be further translated into clinical applications, including noninvasive detection of cancer-related enzyme activity.

Selective determination of cysteines through precolumn double-labeling and liquid chromatography followed by detection of intramolecular FRET

In this paper we introduce a novel approach for highly selective and sensitive analysis of cysteines (glutathione, cysteine, and homocysteine). This method is based on the detection of intramolecular fluorescence resonance energy transfer (FRET) in a liquid chromatography (LC) system after double-labeling of the amino and sulfhydryl groups of the cysteines. In this detection process, we monitored the FRET between the amine-derivatized and thiol-derivatized fluorophores. We screened 16 combinations of fluorescent reagents. As a result, FRET occurred most effectively when the sulfhydryl and amino groups of the cysteines were derivatized with 7-diethylamino-3-[{4'-(iodoacetyl)amino}phenyl]-4-methylcoumarin (DCIA, Ex/Em 390/480 nm) and 4-fluoro-7-nitrobenz-2-oxo-1,3-diazole (NBD-F, Ex/Em 480/540 nm), respectively, in this order. The double-labeled cysteines emitted NBD-F fluorescence (540 nm) through an intramolecular FRET process when they were excited at the wavelength of maximum excitation of DCIA (390 nm). The generation of FRET was confirmed by comparison with analysis of n-amylamine or tryptophan (amines without a sulfhydryl group) and 6-mercaptohexanol (thiol without an amino group) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the double-labeled cysteines (DCIA and NBD-F) when performing LC on an ODS column with isocratic elution. The limits of quantification (signal-to-noise ratio = 10) and detection (signal-to-noise ratio = 3) for the cysteines, for a 20-μL injection volume, were in the range 150-670 fmol and 46-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of a system which takes advantage of conventional detection of the derivatives. Furthermore, this method provides sufficient selectivity and sensitivity to determine the total cysteines present in the plasma of healthy humans.

Strength in Numbers: Effects of Acceptor Abundance on FRET Efficiency

Fluorescence resonance energy transfer (FRET) is a strongly distance-dependent process between a donor and an acceptor molecule, which can be used for sensitive distance measurements and characterization of molecular interactions at the nanometer level. The original mathematical description of this process, however, is only valid for the interaction of one donor with one acceptor. This criterion is not always met, especially in biological systems, where multiple structures can interact simultaneously, often making distance estimations based on transfer efficiency values error-prone. Herein we investigate how the interaction of multiple acceptors and donors influences the transfer efficiency value in an intramolecular cellular FRET system by manipulating the fluorophore/protein ratio of the fluorophore-conjugated antibodies. We show that the labeling ratio of the acceptor has the largest influence on measured transfer efficiency and decreasing or increasing the acceptor labeling ratio can be utilized to manipulate the FRET response of the acceptor-donor pair and therefore is a tool for optimizing sensitivity of FRET measurements.

Intrinsic Properties of immunoglobulin IgG1 Isotype-Switched B Cell Receptors Promote Microclustering and the Initiation of Signaling

Memory B cells express high-affinity, immunoglobulin GB cell receptors (IgG BCRs) that enhance B cell responses, giving rise to the rapid production of high-affinity, IgG antibodies. Despite the central role of IgG BCRs in memory responses, the mechanisms by which the IgG BCRs function to enhance B cell responses are not fully understood. Using high-resolution live-cell imaging, we showed that IgG1 BCRs dramatically enhanced the earliest BCR-intrinsic events that followed within seconds of B cells' encounter with membrane bound antigen, including BCR oligomerization and BCR microcluster growth, leading to Syk kinase recruitment and calcium responses. The enhancement of these early events was dependent on a membrane proximal region of the IgG1 cytoplasmic tail not previously appreciated to play a role in IgG1 BCR signaling. Thus, intrinsic properties of the IgG1 BCR enhance early antigen-driven events that ultimately translate into heightened signaling.

Apparent PKA activity responds to intermittent hypoxia in bone cells: a redox pathway?

We studied hypoxia-induced dynamic changes in the balance between PKA and PKA-counteracting phosphatases in the microfluidic environment in single cells using picosecond fluorescence spectroscopy and intramolecular fluorescence resonance energy transfer (FRET)-based sensors of PKA activity. First, we found that the apparent PKA activity in bone cells (MC3T3-E1 cells) and endothelial cells (bovine aortic endothelial cells) is rapidly and sensitively modulated by the level of O(2) in the media. When the O(2) concentration in the glucose-containing media was lowered due to O(2) consumption by the cells in the microfluidic chamber, the apparent PKA activity increases; the reoxygenation of cells under hypoxia leads to a rapid ( approximately 2 min) decrease of the apparent PKA activity. Second, lack of glucose in the media led to a lower apparent PKA activity and to a reversal of the response of the apparent PKA activity to hypoxia and reoxygenation. Third, the apparent PKA activity in cells under hypoxia was predominantly regulated via a cAMP-independent pathway since 1) changes in the cAMP level in the cells were not detected using a cAMP FRET sensor, 2) the decay of cAMP levels was too slow to account for the fast decrease in PKA activity levels in response to reoxygenation, and 3) the response of the apparent PKA activity due to hypoxia/reoxygenation was not affected by an adenylate cyclase inhibitor (MDL-12,330A) at 1 mM concentration. Fourth, the immediate onset of ROS accumulation in MC3T3-E1 cells subjected to hypoxia and the sensitivity of the apparent PKA activity to redox levels suggest that the apparent PKA activity change during hypoxia and reoxygenation in this study can be linked to a redox potential change in response to intermittent hypoxia through the regulation of activities of PKA-counteracting phosphatases such as protein phosphatase 1. Finally, our results suggest that the detection of PKA activity could be used to monitor responses of cells to hypoxia in real time.

Polarization of Migrating Monocytic Cells Is Independent of PI 3-Kinase Activity

BackgroundMigration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization.Methodology/Principal FindingsWe present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the α2A-adrenoceptor (α2AAR) display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homlogue UK 14'304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microcopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET) of α2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinse activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes – in contrast to neutrophils – rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation.Conclusions/SignificanceAccumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process appears to be independent of PI 3-kinase activity.

Poly(p‐phenylenevinylene) Derivatives with Defined Conjugation Segments and Post‐Polymerization Modification with Sterically Enshrouded Chromophores

Two poly(p-phenylenevinylene) derivative alternating copolymers (P1-I and P2-I) have been prepared featuring iodo substituents and m-phenylene units to periodically disrupt conjugation. P1-I was derivatized with various chromophores to yield P1a-f. In P1a-f, the chromophores were positioned within a sterically protected pocket shielding them from interchain interactions so that intrachain interactions between polymer segments could be observed. Solution and film properties of polymers have been examined. Post-polymerization chromophore modification leads to new photophysical properties such as intramolecular charge transfer and fluorescent resonance energy transfer processes in some cases.

Efficient Fluorescence Resonance Energy Transfer-Based Ratiometric Fluorescent Cellular Imaging Probe for Zn2+ Using a Rhodamine Spirolactam as a Trigger

This letter described the design and synthesis of a novel fluorescein-appended rhodamine spirolactam derivative and its preliminary application as a ratiometric fluorescent cellular imaging probe for Zn(2+). The ratiometric fluorescent signal change of the probe is based on an intramolecular fluorescence resonance energy transfer (FRET) mechanism modulated by a specific metal ion induced ring-opening process of the rhodamine spirolactam (acting as a trigger). In the new developed sensing system, the emission peaks of the two fluorophores are well-resolved, which can avoid the emission spectra overlap problem generally met by spectra-shift type probes and benefits for observation of fluorescence signal change at two different emission wavelengths with high resolution. It also benefits for a large range of emission ratios, thereby a high sensitivity for Zn(2+)detection. Under optimized experimental conditions, the probe exhibits a stable response for Zn(2+) over a concentration range from 2.0 x 10(-7) to 2.0 x 10(-5) M, with a detection limit of 4.0 x 10(-8) M. Most importantly, the novel probe has well solved the problem of serious interferences from other transition metal ions generally met by previously reported typical fluorescent probes for Zn(2+) with the di(2-picolyl)amine moiety as the receptor (in this case, the fluorescence response induced by Cd(2+)is even comparable to that of Zn(2+)) and shows a reversible and fast response toward Zn(2+). All these unique features make it particularly favorable for ratiometric cellular imaging investigations. It has been preliminarily used for ratiometric imaging of Zn(2+) in living cells with satisfying resolution.

2-Aminotryptanthrin Derivative with Pyrene as a FRET-based Fluorescent Chemosensor for Al3+

1-(2-Tryptanthrinylaminoacetoxy)-14-(1-pyrenecarboxy)-3,6,9,12-tetraoxatetradecane (T2NH-P5P) was synthesized as a fluorescent chemosensor for Al3+. Excited at 325 nm, corresponding to absorption of the pyrene unit of T2NH-P5P, emission at 600 nm from the 2-aminotryptanthrin unit has been observed, indicating that intramolecular fluorescence resonance energy transfer (FRET) occurs in T2NH-P5P. However, when Al3+ is added to a solution of T2NH-P5P, the fluorescence of 2-aminotryptanthrin is quenched (FRET-off), whereas that of the pyrene group is revived. Such a FRET "on-off" behavior of T2NH-P5P is not observed for other metal cations (Ca2+, Ba2+, and Zn2+).

Fibronectin forms the most extensible biological fibers displaying switchable force-exposed cryptic binding sites

Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stress-strain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed.

Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors

Small GTPases act as molecular switches that regulate a variety of cellular functions, such as proliferation, cell movement and vesicle trafficking. Genetically encoded biosensors based on the principle of fluorescence resonance energy transfer (FRET) can visualize a spatio-temporal activity of small GTPases in living cells, thereby helping us to understand the role of small GTPases intuitively and vividly. Here we describe protocols of live cell imaging with the FRET biosensors. There are several types of FRET biosensors; this protocol focuses on intramolecular or unimolecular FRET biosensors of small GTPases that are made up of donor and acceptor fluorescence proteins, a small GTPase, its binding partner, and, if necessary, a subcellular localization signal. These FRET biosensors uncover the spatio-temporal activity of the small GTPases in living cells, which could not be obtained by conventional biochemical methods. Preparation of FRET biosensors and cell culture takes 6 d. Imaging and processing take 3-4 d to complete.