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PubMed2005

Highlighting protein kinase CK2 movement in living cells.

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (alpha2beta2) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic alpha and regulatory beta subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol 23: 975-987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio-temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.

3T3 Cells↗

PubMed1989

Role of corrosion in Harrington and Luque rods failure.

Ten in-vivo failed spinal instrumentation systems, i.e. six Harrington distraction rods with sublaminar hooks, one Harrington distraction rod with segmental wiring and three Luque rods with sublaminar wires, were fractographically analysed. In both Harrington and Luque rods corrosion fatigue was the predominant mechanism resulting in the failure. Five Harrington rods fractured at the first ratchet junction; fractures of the Luque rods were initiated by fretting of sublaminar wire with the rod surface in the presence of spinal non-union. Fretting and crevice corrosion were found to play an important role in compromising the segmental spinal instrumentation. The susceptibility of the 316 L CW austenitic stainless steel to pitting and relative resistance to crevice corrosion were measured by cyclic anodic polarization tests. The oxide inclusions have been found to play a significant role in the pitting of the alloy.

Corrosion↗

PubMed1996

Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein.

We report fluorescent resonance energy transfer (FRET) between two linked variants of the green fluorescent protein (GFP). The C terminus of a red-shifted variant of GFP (RSGFP4) is fused to a flexible polypeptide linker containing a Factor X a protease cleavage site. The C terminus of this linker is in turn fused to the N terminus of a blue variant of GFP (BFP5). The gene product has spectral properties that suggest energy transfer is occurring from BFP5 to RSGFP4. Upon incubation with Factor X(a), the protein is cleaved, and the two fluorescent proteins dissociate. This is accompanied by a marked decrease in energy transfer. The RSGFP4::BFP5 fusion protein demonstrates the feasibility of using FRET between two GFP derivatives as a tool to monitor protein-protein interactions; in addition, this construct may find applications as an intracellular screen for protease inhibitors.

Animals↗

PubMed2004

A homogeneous time-resolved fluorescence detection of telomerase activity.

The homogeneous time-resolved fluorescence (HTRF) technology is an assay developed to study the interaction between biomolecules. This detection system is based on a fluorescence resonance energy transfer (FRET) between a Tris-bipyridine europium cryptate used as a long-lived fluorescent donor and a chemically modified allophycocyanine as acceptor. This technology is characterized by both a spectral selectivity and a temporal selectivity (due to the time-resolved mode), ensuring a highly specific signal. Here a europium-cryptate-labeled deoxyuridine triphosphate analogue (K-11-dUTP) was used to monitor the extension reaction on a biotinylated oligonucleotide used as substrate for telomerase in a telomeric repeat amplification protocol (TRAP). After the addition of an allophycocyanine-streptavidin conjugate, the extension products give rise to a FRET between the incorporated cryptate moieties and the allophycocyanine acceptor that then displays a specific long-lived emission. The TRAP-HTRF format was validated as a screening tool by using a 2,6-diaminoanthraquinone analogue, a known inhibitor of telomerase activity. The IC(50) measured was consistent with the reported values, showing the convenience of the HTRF technology for the study of telomerase activity and inhibitors.

Anthraquinones↗

PubMed2004

Fluorescence resonance energy transfer-based detection of analytes using antiidiotypic affinity protein pairs.

A new method for specific detection of proteins based on fluorescence resonance energy transfer (FRET) using affinity proteins (affibodies) derived from combinatorial engineering of Staphylococcal protein A has been developed. Antiidiotypic affibody pairs were used in a homogeneous competitive binding assay, where the idiotypic, target-specific affibody was labeled with fluorescein and the antiidiotypic affibody was labeled with tetramethylrhodamine. Intermolecular FRET between the two fluorescent probes was observed in the antiidiotypic affibody complex, but upon addition of target protein the antiidiotypic affibody was displaced, which was monitored by a shift in the relative emission of the donor and acceptor fluorophores. The feasibility of the system was demonstrated by the detection of IgA and Taq DNA polymerase with high specificity, using two different antiidiotypic affibody pairs. Detection of Taq DNA polymerase in 25% human plasma was successfully carried out, demonstrating that the method can be used for analysis of proteins in samples of complex composition.

Binding Sites↗

PubMed2005

A homogeneous, high-throughput fluorescence resonance energy transfer-based DNA polymerase assay.

A homogeneous, fluorescence resonance energy transfer (FRET)-based DNA polymerase assay that is suitable for high-throughput screening for inhibitors, and can also be used for steady-state kinetic investigations, is described. The activity, kinetic mechanism, and processivity of the isolated alpha subunit of DNA polymerase III, the product of the dnaE gene, from the gram-negative pathogen Haemophilus influenzae were investigated using the FRET assay.

Base Sequence↗

PubMed2006

Development of homogeneous binding assays based on fluorescence resonance energy transfer between quantum dots and Alexa Fluor fluorophores.

We studied the fluorescence resonance energy transfer (FRET) between quantum dots emitting at 565, 605, and 655 nm as energy donors and Alexa Fluor fluorophores with absorbance maxima at 594, 633, 647, and 680 nm as energy acceptors. As a first step, we prepared covalent conjugates between all three types of quantum dots and each of the Alexa Fluor fluorophores that could act as an energy acceptor. All of these conjugates displayed efficient resonance energy transfer. Then we prepared covalent conjugates of these quantum dots with biotin, fluorescein, and cortisol and established that the binding of these conjugates to suitable Alexa Fluor-labeled antibodies and streptavidin (in the case of biotin) can be efficiently detected by measuring the resonance energy transfer in homogeneous solutions. Finally, based on these observations, competitive binding assays for these three small analytes were developed. The performance of these assays as a function of the degree of labeling of the quantum dots was evaluated. It was found that decreasing the degree of loading of the quantum dots leads to decreases of the limits of detection. The results show the great potential of this FRET system for the development of new homogeneous binding assays.

Binding, Competitive↗

PubMed2006

Enhanced fluorescence resonance energy transfer immunoassay with improved sensitivity based on the Fab'-based immunoconjugates.

Fluorescence resonance energy transfer (FRET) is a powerful technique to monitor protein-protein interaction. Recently, we developed homogeneous and noncompetitive immunoassay based on the enhanced FRET by leucine zipper interaction. Here we improved the assay by establishing a general method for preparation of the Fab'-based immunoconjugate. Anti-human serum albumin Fab' numbers 11 and 13 were chemically conjugated with recombinant proteins consisting of thioredoxin, flexible linker, and green fluorescent protein color variant tethered with a leucine zipper motif. Compared with single chain antibody variable region-based fusion proteins prepared by the gene fusion method in our previous study, the resultant Fab'-based immunoconjugates accomplished an assay with nearly 10 times greater sensitivity. Furthermore, the conjugation method enabled us to apply the assay generally to measurement of another high-molecular weight antigen for which antibodies prepared for sandwich immunoassay are commercially available. Because of the facility and generality of the preparation method for the immunoconjugate, the assay is expected to be applied to many antigens that require rapid diagnosis and moderate measurement range.

Fluorescence Resonance Energy Transfer↗

PubMed2004

Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer.

Mitochondrial depolarization promotes apoptotic and necrotic cell death and possibly other cellular events. Polarized mitochondria take up cationic tetramethylrhodamine methylester (TMRM), which is released after depolarization. Thus, TMRM does not label depolarized mitochondria. To identify both polarized and depolarized mitochondria in living cells, cultured rat hepatocytes, and sinusoidal endothelial cells were co-loaded with green-fluorescing MitoTracker Green FM (MTG) and red-fluorescing TMRM for imaging by laser scanning confocal microscopy. Like TMRM, MTG is a cationic fluorophore that accumulates electrophoretically into polarized mitochondria. Unlike TMRM, MTG binds covalently to intramitochondrial protein thiols and remains bound after depolarization. In cells labeled only with MTG, excitation with blue (488 nm) light yielded green but almost no red fluorescence. After subsequent loading with TMRM, green MTG fluorescence became quenched. Instead, blue excitation yielded red fluorescence. Mitochondrial de-energization restored green fluorescence and abolished red fluorescence. Conversely, when MTG was added to TMRM-labeled cells, red fluorescence excited by blue light was enhanced, an effect again reversed by de-energization. These observations of reversible quenching of donor fluorescence and augmentation of acceptor fluorescence signify fluorescence resonance energy transfer (FRET). In undisturbed hepatocytes, spontaneous depolarization of a subfraction of mitochondria was an ongoing phenomenon. In conclusion, confocal FRET discriminates individual depolarized mitochondria against a background of hundreds of polarized mitochondria.

Aldehydes↗

PubMed2005

Oligomeric behavior of Bordetella pertussis adenylate cyclase toxin in solution.

Adenylate cyclase (AC) toxin from Bordetella pertussis inserts into eukaryotic cells, producing intracellular cAMP, as well as hemolysis and cytotoxicity. Concentration dependence of hemolysis suggests oligomers as the functional unit and inactive deletion mutants permit partial restoration of intoxication and/or hemolysis, when added in pairs [M. Iwaki, A. Ullmann, P. Sebo, Mol. Microbiol. 17 (1995) 1015-1024], suggesting dimerization/oligomerization. Using affinity co-precipitation and fluorescence resonance energy transfer (FRET), we demonstrate specific self-association of AC toxin molecules in solution. Flag-tagged AC toxin mixed with biotinylated-AC toxin, followed by streptavidin beads, yields both forms of the toxin. FRET measurements of toxin, labeled with different fluorophores, demonstrate association in solution, requiring post-translational acylation, but not calcium. AC toxin mixed with DeltaR, an inactive mutant, results in enhancement of hemolysis over that with wild type alone, suggesting that oligomers are functional. Dimers and perhaps higher molecular mass forms of AC toxin occur in solution in a manner that is relevant to toxin action.

Adenylate Cyclase Toxin↗

PubMed2006

Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament.

Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnI). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a beta-turn/coil motif to an extended conformation upon Ca(2+) activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca(2+)-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnI(L129W/S151C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor-acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca(2+)-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca(2+) titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin S1 to AEDANS attached to Cys151 of cTnI was very small and Ca(2+) independent. The present results show that the rate of Ca(2+)-induced structural transition and Ca(2+) sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca(2+) sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn-cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound S1 significantly increased the Ca(2+) sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca(2+) sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by beta-adrenergic stimulation.

Binding Sites↗

PubMed2005

Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein.

Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, approximately 20 A from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus.

Amino Acid Sequence↗

PubMed2005

Functional co-localization of monocytic aminopeptidase N/CD13 with the Fc gamma receptors CD32 and CD64.

Information about the function of aminopeptidase N/CD13 on monocytes is limited. In order to gain more insight into its interaction with other proteins, we have identified molecules that co-localize with the membrane ectoenzyme at the cell surface of monocytes. Using laser scanning and electron microscopy as well as fluorescence resonance energy transfer (FRET) measured by flow cytometry we show that monocytic CD13 co-localized with the Fc gamma receptor II/CD32 after Fc receptor ligation by a CD32-specific antibody. FRET was also observed between CD13 and the Fc gamma receptor I/CD64, but not with the myeloid marker CD33 representing a member of the sialoadhesin family. Our results imply a novel functional role of CD13 and Fc gamma receptors as members of a multimeric receptor complex. Further studies have to be done to elucidate common signaling pathways of these molecules.

CD13 Antigens↗

PubMed2006

The N-terminal domain of tumor suppressor p53 is involved in the molecular interaction with the anti-apoptotic protein Bcl-Xl.

Emerging evidences suggest that transcription-independent mechanism of p53 appears to make an important contribution to the overall p53-dependent apoptosis. Recently, it has been postulated that the DNA-binding domain of p53 can interact with Bcl-Xl, and subsequently the proposed molecular interaction has been shown by NMR studies. Interestingly, Chipuk et al. [Cancer Cell 4 (2003) 371] reported that the N-terminal domain of p53 (p53NTD) alone is necessary and sufficient to induce transcription-independent apoptosis. To further define and understand the nature of the molecular recognition between p53 and Bcl-Xl, our current study focuses on p53NTD. We first demonstrated the molecular interaction between p53NTD and Bcl-Xl by co-expressing and purifying the complex. Second, to define the binding interface of the molecular interaction, which is not previously characterized, in the current we employed a NMR-based binding study, showing that the binding site on Bcl-Xl is located in the region including alpha4, the N- and C-termini of alpha3, the N-terminus of alpha5, and the central part of alpha2. To further probe this observation, we then performed fluorescence resonance energy transfer (FRET) assay in cells. The FRET efficiency detected between the donor and acceptor molecules appears to suggest the presence of molecular interaction of p53NTD with Bcl-Xl in cells. Taken together, our data suggest that p53NTD interacts with Bcl-Xl but the characteristic of the molecular interaction appears to be different from that of the DNA-binding domain of p53.

Cell Line, Tumor↗

PubMed2006

A self-assembled quantum dot probe for detecting beta-lactamase activity.

This communication describes a quantum dot probe that can be activated by a reporter enzyme, beta-lactamase. Our design is based on the principle of fluorescence resonance energy transfer (FRET). A biotinylated beta-lactamase substrate was labeled with a carbocyanine dye, Cy5, and immobilized on the surface of quantum dots through the binding of biotin to streptavidin pre-coated on the quantum dots. In assembling this nanoprobe, we have found that both the distance between substrates and the quantum dot surface, and the density of substrates are important for its function. The fluorescence emission from quantum dots can be efficiently quenched (up to 95%) by Cy5 due to FRET. Our final quantum dot probe, assembled with QD605 and 1:1 mixture of biotin and a Cy5-labeled lactam, can be activated by 32microg/mL of beta-lactamase with 4-fold increase in the fluorescence emission.

Fluorescence Resonance Energy Transfer↗

PubMed2006

TRAIL-induced apoptosis proceeding from caspase-3-dependent and -independent pathways in distinct HeLa cells.

The apoptotic pathway in higher eukaryotes remains controversial with respect to the necessity of activation of caspase-3 in TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-treated cells. In this study, a fluorescence resonance energy transfer (FRET) probe was developed to image the activation of caspase-3 and the related apoptotic pathway in TRAIL-treated cells in real time. Both kinds of apoptotic pathways were observed simultaneously in the same experiment proceeding from activation and non-activation of caspase-3. The total apoptotic rate was 56.08%, the apoptotic rates for activation and non-activation of caspase-3 pathways were 21.5% and 34.58%, respectively, which were examined later for Hoechst 33258 staining and morphological characteristics. The apoptotic rate due to the activation of caspase-3 pathways in TRAIL-treated cells has been independently measured to be around 25.11% by capillary electrophoresis (CE) analysis, which confirmed the apoptotic rate due to activation of caspase-3 pathways as found by FRET analysis. This result also suggests that rest apoptosis is preceded by caspase-3-independent pathways, as CE has the ability to quantitatively detect caspase-dependent apoptosis. The observation of the coexistence of caspase-3-dependent and caspase-3-independent apoptotic pathways in the TRAIL-treated cells was unusual in comparison with the previous reports.

Apoptosis↗

PubMed2006

Detection of constitutive heterodimerization of the integrin Mac-1 subunits by fluorescence resonance energy transfer in living cells.

Macrophage differentiation antigen associated with complement three receptor function (Mac-1) belongs to beta2 subfamily of integrins that mediate important cell-cell and cell-extracellular matrix interactions. Biochemical studies have indicated that Mac-1 is a constitutive heterodimer in vitro. Here, we detected the heterodimerization of Mac-1 subunits in living cells by means of two fluorescence resonance energy transfer (FRET) techniques (fluorescence microscopy and fluorescence spectroscopy) and our results demonstrated that there is constitutive heterodimerization of the Mac-1 subunits and this constitutive heterodimerization of the Mac-1 subunits is cell-type independent. Through FRET imaging, we found that heterodimers of Mac-1 mainly localized in plasma membrane, perinuclear, and Golgi area in living cells. Furthermore, through analysis of the estimated physical distances between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to Mac-1 subunits, we suggested that the conformation of Mac-1 subunits is not affected by the fusion of CFP or YFP and inferred that Mac-1 subunits take different conformation when expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293T cells, respectively.

Animals↗

PubMed2006

Three-stage refolding/unfolding of the dual-color beta-subunit in R-phycocyanin from Polysiphonia urceolata.

The conformational changes during refolding and unfolding of the dual-color beta-subunit in R-phycocyanin (R-PC) were monitored by the spectra, fluorescence anisotropy, and FRET. It was observed that both of the refolding and unfolding of the beta-subunit would undergo a three-stage conformational change, but in a reverse order. During the refolding process, at the first stage, the configuration of the tetrapyrrole chromophores transformed from the cyclohelical to the extended one, suggested by the blue-shifted spectra. At the second stage, recovery of the hydrogen-bond and hydrophobic interaction network fixed the chromophore in a more rigid configuration, suggested by a linear increase in the total fluorescence yield. At the third stage, the increase of the FRET efficiency suggested a protein-framework movement that made the two chromophores closer or/and into a more parallel orientation. The fluorescence anisotropy further confirmed the three-stage model.

Fluorescence Resonance Energy Transfer↗