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At least 1,009 records · Page 56Linked to original sources
PubMed1999

Analysis of vertical fluorescence resonance energy transfer from the surface of a small-diameter sphere.

Fluorescence resonance energy transfer (FRET) measurements have been used to analyze fluorophore separations in a number of varying geometries, including small particles and extended surfaces. This study focuses on the geometry created by a donor extended above the surface of a small sphere (radius < R0), where the acceptors are integrated into the sphere surface. The model of this geometry was based on an amphipathic molecule with its lipophilic region integrated into a detergent micelle and its hydrophilic region extending outward from the micelle surface, where the donor fluorophore is attached to the hydrophilic region of the molecule. Based on random acceptor incorporation into the micelle, a Poisson distribution was used to calculate the distribution of acceptor probes across the micelle population. The model converges to RET on a flat surface when the radius of the micelle exceeds 0.8 R0. The model was also used to simulate FRET data showing that the positions of donors above the micelle surface could be uniquely resolved. Experimental verification of the model was achieved in a sulfobetaine palmitate micelle with fluorescein isothiocyanate donors attached to detergent-solubilized lipopolysaccharide (LPS) and lipophilic Fast-DiI acceptors. The use of steady-state analysis allowed resolution of cases in which donors were located at different distances from the surface. Combining steady-state with excited-state lifetime analysis allowed resolution of cases where there was a combination of distances. Given the large number of biomolecules that interact with lipids, this approach may prove generally useful for defining molecular conformation.

Biophysical Phenomena↗
PubMed1999

Scanning near-field fluorescence resonance energy transfer microscopy.

A new microscopic technique is demonstrated that combines attributes from both near-field scanning optical microscopy (NSOM) and fluorescence resonance energy transfer (FRET). The method relies on attaching the acceptor dye of a FRET pair to the end of a near-field fiber optic probe. Light exiting the NSOM probe, which is nonresonant with the acceptor dye, excites the donor dye introduced into a sample. As the tip approaches the sample containing the donor dye, energy transfer from the excited donor to the tip-bound acceptor produces a red-shifted fluorescence. By monitoring this red-shifted acceptor emission, a dramatic reduction in the sample volume probed by the uncoated NSOM tip is observed. This technique is demonstrated by imaging the fluorescence from a multilayer film created using the Langmuir-Blodgett (LB) technique. The film consists of L-alpha-dipalmitoylphosphatidylcholine (DPPC) monolayers containing the donor dye, fluorescein, separated by a spacer group of three arachidic acid layers. A DPPC monolayer containing the acceptor dye, rhodamine, was also transferred onto an NSOM tip using the LB technique. Using this modified probe, fluorescence images of the multilayer film reveal distinct differences between images collected monitoring either the donor or acceptor emission. The latter results from energy transfer from the sample to the NSOM probe. This method is shown to provide enhanced depth sensitivity in fluorescence measurements, which may be particularly informative in studies on thick specimens such as cells. The technique also provides a mechanism for obtaining high spatial resolution without the need for a metal coating around the NSOM probe and should work equally well with nonwaveguide probes such as atomic force microscopy tips. This may lead to dramatically improved spatial resolution in fluorescence imaging.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Agonist-independent and -dependent oligomerization of dopamine D(2) receptors by fusion to fluorescent proteins.

Oligomerization of the short (D(2S)) and long (D(2L)) isoforms of the dopamine D(2) receptor was explored in transfected Cos-7 cells by their C-terminal fusion to either an enhanced cyan or enhanced yellow fluorescent protein (ECFP or EYFP) and the fluorescent fusion protein interaction was monitored by a fluorescence resonance energy transfer (FRET) assay. The pharmacological properties of the fluorescent fusion proteins, as measured by both displacement of [(3)H]nemonapride binding and agonist-mediated stimulation of [(35)S]GTPgammaS binding upon co-expression with a G(alphao)Cys(351)Ile protein, were not different from the respective wild-type D(2S) and D(2L) receptors. Co-expression of D2S:ECFP+D2S:EYFP in a 1:1 ratio and D2L:ECFP+D2L:EYFP in a 27:1 ratio resulted, respectively, in an increase of 26% and 16% in the EYFP-specific fluorescent signal. These data are consistent with a close proximity of both D(2S) and D(2L) receptor pairs of fluorescent fusion proteins in the absence of ligand. The agonist-independent D(2S) receptor oligomerization could be attenuated by co-expression with either a wild-type, non-fluorescent D(2S) or D(2L) receptor subtype, but not with a distinct beta(2)-adrenoceptor. Incubation with the agonist (-)-norpropylapomorphine dose-dependently (EC(50): 0.23+/-0.06 nM) increased the FRET signal for the co-expression of D2S:ECFP and D2S:EYFP, in support of agonist-dependent D(2S) receptor oligomerization. In conclusion, our data strongly suggest the occurrence of dopamine D(2) receptor oligomers in intact Cos-7 cells.

Animals↗
PubMed2002

Stepwise rotation of the gamma-subunit of EF(0)F(1)-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer.

The EF(0)F(1)-ATP synthase mutants bQ64C and gamma T106C were labelled selectively with the fluorophores tetramethylrhodamine (TMR) at the b-subunit and with a cyanine (Cy5) at the gamma-subunit. After reconstitution into liposomes, these double-labelled enzymes catalyzed ATP synthesis at a rate of 33 s(-1). Fluorescence of TMR and Cy5 was measured with a confocal set-up for single-molecule detection. Photon bursts were detected, when liposomes containing one enzyme traversed the confocal volume. Three states with different fluorescence resonance energy transfer (FRET) efficiencies were observed. In the presence of ATP, repeating sequences of those three FRET-states were identified, indicating stepwise rotation of the gamma-subunit of EF(0)F(1).

Adenosine Triphosphate↗
PubMed2002

Elucidating a key intermediate in homologous DNA strand exchange: structural characterization of the RecA-triple-stranded DNA complex using fluorescence resonance energy transfer.

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA-tsDNA complex), has been reported. We present a systematic characterization of the helical geometries of the three DNA strands of the RecA-tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. FRET donor and acceptor dyes were used to label different DNA strands, and the interfluorophore distances were inferred from energy transfer efficiencies measured as a function of the base-pair separation between the two dyes. The energy transfer efficiencies were first measured on a control RecA-dsDNA complex, and the calculated helical parameters (h approximately 5 A, Omega(h) approximately 20 degrees ) were consistent with structural conclusions derived from electron microscopy (EM) and other classic biochemical methods. Measurements of the helical parameters for the RecA-tsDNA complex revealed that all three DNA strands adopt extended and unwound conformations similar to those of RecA-bound dsDNA. The structural data are consistent with the hypothesis that this complex is a late, post-strand-exchange intermediate with the outgoing strand shifted by about three base-pairs with respect to its registry with the incoming and complementary strands. Furthermore, the bases of the incoming and complementary strands are displaced away from the helix axis toward the minor groove of the heteroduplex, and the bases of the outgoing strand lie in the major groove of the heteroduplex. We present a model for the strand exchange intermediate in which homologous contacts preceding strand exchange arise in the minor groove of the substrate dsDNA.

Adenosine Triphosphate↗
PubMed2004

Fluorescence-based assays for RGS box function.

Ligand-activated, seven transmembrane-spanning receptors interact with inactive G-protein heterotrimers (Galphabetagamma) to catalyze GTP loading and, consequently, activation of Galpha subunits and the liberation of Gbetagamma. Galpha.GTP and Gbetagamma are then competent to regulate independent effector pathways. The duration of heterotrimeric G-protein signaling is determined by the lifetime of the Galpha subunit in the GTP-bound state. Signal termination is facilitated by the intrinsic guanosine triphosphatase (GTPase) activity of Galpha and subsequent reformation of the inactive heterotrimer. Regulators of G-protein signaling (RGS) proteins act enzymatically, via their hallmark "RGS box," as GTPase-accelerating proteins (GAPs) for Galpha subunits and thus function as negative regulators of G-protein signaling in vitro and in vivo. This article describes the use of fluorescence resonance energy transfer (FRET) to monitor the interaction between a Galpha subunit and an RGS box protein. Furthermore, this article describes optimization of this assay for high-throughput screening and the evaluation of mutant RGS box and Galpha proteins. Finally, this article describes the novel application of this FRET technique to measure the activity of RGS protein-derived GoLoco peptides that modulate Galpha activation by aluminum tetrafluoride.

Amino Acid Substitution↗
PubMed2005

Reconstitution of cargo-dependent COPII coat assembly on proteoliposomes.

The coat protein complex II (COPII) coat is responsible for direct capture of membrane cargo proteins and for the physical deformation of the endoplasmic reticulum (ER) membrane that drives the transport vesicle formation. The use of an in vitro reconstitution system comprising purified components is desirable for studies aimed at elucidating the role(s) of individual proteins in a specific biochemical reaction. To investigate the assembly-disassembly of COPII coats in a completely reconstituted reaction, we have developed a synthetic budding reaction involving purified coat proteins and cargo-reconstituted proteoliposomes. We describe here a fluorescence resonance energy transfer (FRET)-based method for monitoring the kinetics of COPII coat complex assembly and disassembly in cargo-reconstituted proteoliposomes. This assay allows comparison of the time course of the coat disassembly from the cargo as monitored by FRET signal with the time course of accompanying Sar1p GTP hydrolysis by tryptophan fluorescence.

COP-Coated Vesicles↗
PubMed2006

Analysis of Ras activation in living cells with GFP-RBD.

Several genetically encoded fluorescent biosensors for Ras family GTPases have been developed that permit spatiotemporal analysis of the activation of these signaling molecules in living cells. We describe here the use of the simplest of these probes, the Ras binding domain (RBD) of selected effectors fused with green fluorescent protein (GFP) or one of its spectral mutants. When expressed in quiescent cells, these probes are distributed homogeneously through the cytosol and nucleoplasm. On activation of their cognate GTPases on membranes, they are recruited to these compartments, and activation can be scored by redistribution of the probe. The advantage of this system is its simplicity: the probes are genetically encoded and can easily be constructed with standard cloning techniques, and the readout of activation requires only standard epifluorescence or confocal microscopy. The disadvantage of the system is that only rarely are Ras-related GTPases expressed at high enough levels to permit detection of the activation of the endogenous proteins. In general, the method requires overexpressing untagged, wild-type versions of the GTPase of interest. However, we describe a FRET-based method called bystander FRET developed to detect endogenous proteins that can be used to validate the results obtained by overexpressing Ras proteins. By use of this technique, we and others have uncovered important new features of the spatiotemporal regulation of Ras and related GTPases.

Animals↗
PubMed2006

Activation of Rap1, Cdc42, and rac by nectin adhesion system.

Nectin is an immunoglobulin-like cell-cell adhesion molecule and forms adherens junctions cooperatively with cadherin. Trans-interaction of nectin induces activation of Rap1, Cdc42, and Rac small G proteins. The activity of these small G proteins can be analyzed by the pull-down assay using GST-Ral-GDS fusion protein for Rap1 and GST-PAK-CRIB for Cdc42 and Rac. The fluorescent resonance energy transfer (FRET) system is also available to spatially and temporally detect the activity of these small G proteins in the living cells. In addition to these assays, the activity of Cdc42 and Rac is indirectly, but easily, evaluated by the cell-spreading assay to examine formation of filopodia and lamellipodia, respectively. To clearly explore the effect of trans-interacting nectin on the small G proteins, we use L fibroblasts stably expressing nectin-1 (nectin-1-L cells) and the extracellular domain of nectin-3 fused to human IgG Fc (Nef-3). Treatment of nectin-1-L cells with Nef-3 remarkably increases both the amount of the GTP-bound form and the FRET efficiency of all Rap1, Cdc42, and Rac small G proteins. In the cell-spreading assay, Cdc42 and Rac activated in this way promote the formation of filopodia and lamellipodia, respectively. Here, we focus on how the activity of Cdc42 and Rac induced by trans-interacting nectin is examined by use of the pull-down and the cell-spreading assays.

Animals↗
PubMed2004

Structural basis for FEN-1 substrate specificity and PCNA-mediated activation in DNA replication and repair.

Flap EndoNuclease-1 (FEN-1) and the processivity factor proliferating cell nuclear antigen (PCNA) are central to DNA replication and repair. To clarify the molecular basis of FEN-1 specificity and PCNA activation, we report here structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular beta sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. The DNA and protein conformational changes, composite complex structures, FRET, and mutational results support enzyme-PCNA alignments and a kinked DNA pivot point that appear suitable to coordinate rotary handoffs of kinked DNA intermediates among enzymes localized by the three PCNA binding sites.

Archaeoglobus↗
PubMed1999

Surface topography, corrosion and microhardness of nitrogen-diffusion-hardened titanium alloy.

Mechanical-electrochemical interactions accelerate corrosion in mixed-metal modular hip prostheses. These interactions can be reduced by improving the modular component machining tolerances or by improving the resistance of the components to scratch or fretting damage. Wrought cobalt-alloy (CoCrMo) is known to have better tribological properties compared to the titanium alloy (Ti64). Thus, improving the tribological properties of this mixed-metal interface should center around improving the tribological properties of the Ti64 alloy. This study used scanning probe microscopy (contact, tapping and phase contrast mode), scanning electron microscopy, corrosion testing, and microhardness testing to determine the effect of a nitrogen-diffusion hardening process on the surface morphology, electrochemistry and surface hardness of the Ti64 alloy. The nitrogen-diffusion-hardened titanium alloy samples (N-Ti64) had a more pronounced grain structure, more nodular surface, and significantly (P<0.01) higher mean roughness values than the control-Ti64 samples. The N-Ti64 samples also exhibited at least equivalent corrosion behavior and a definite increase in surface hardness compared to the control Ti64 samples. The equivalent corrosion behavior and improved surface hardness indicate the potential for N-Ti64 samples to resist similar and mixed-metal scratch and fretting damage. The use of N-Ti64 as opposed to control-Ti64 may therefore reduce the occurrence of mechanical-electrochemical degradation in mixed-metal modular total hip prostheses.

Alloys↗
PubMed2000

The in-vivo wear performance of prosthetic femoral heads with titanium nitride coating.

This paper reports the study performed on four titanium nitride (TiN) coated prosthetic femoral heads collected at revision surgery together with patient data. Surface topology has been examined using Scanning Electron Microscopy (SEM) and elemental analysis of both coating and substrate have been evaluated using energy-dispersive X-ray spectrometry. Quantitative assessment of the surface topography is achieved using contacting profilometry. The average Ra roughness value is calculated at five different locations for each femoral head. The UHMWPE counterface worn volume has been measured directly on the acetabular components. TiN fretting and coating breakthrough occurred in two of the four components examined. In the damaged coating areas the surface profile is macroscopically saw-toothed with average tooth height 1.5 microm. The average Ra value is 0.02 microm on the undamaged surfaces and 0.37 microm on the damaged ones. Failure of the coating adhesion resulted in the release of TiN fragments and of metallic particulate from the substrate fretting corrosion and in the increase of the head surface roughness affecting counterface debris production. Our results suggest that TiN-coated titanium alloy femoral heads are inadequate in the task of resisting third body wear mechanisms in vivo.

Biocompatible Materials↗
PubMed2003

Allogeneic and syngeneic class I MHC complexes drive the association of CD8 and TCR on 2C T cells.

In most cases, cytotoxic T cell activation is dependent on the interaction of the T cell receptor (TCR) and CD8 with MHC class I molecules. In the CD8(+) T cell system based on the mouse cytotoxic T cell clone 2C, recognition of the allogeneic MHC L(d) exhibits a less significant role for CD8 than recognition of the syngeneic MHC K(b). Here, we examined whether this difference is related to the relative abilities of the two pepMHC complexes to drive the association of CD8 and TCR on the T cell surface. We show that both the syngeneic and allogeneic pepMHC induced association of CD8 and TCR, as revealed by fluorescence resonance energy transfer (FRET). Thus, the orientation of the syngeneic and allogeneic ligands when bound to the same TCR both allow CD8 to be recruited to the TCR complex. The conserved diagonal orientation of TCRs on different pepMHC ligands may facilitate such associations. The FRET results are consistent with the known binding properties and the CD8 involvement of the two different TCR:pepMHC interactions.

Animals↗
PubMed2003

A convenient approach to the generation of multiple internal control DNA for a panel of real-time PCR assays.

Real-time polymerase chain reaction (PCR) assays allow convenient detection and quantitation of virus-derived nucleic acids in clinical specimens. When specimens are assayed for the presence of virus-derived nucleic acids against external standards, sample adequacy is not monitored. This can be achieved by using internal controls that are co-amplified with the virus-specific DNA in competitive PCR. Each of the various real-time PCR assays in a routine clinical laboratory requires its specific internal control. In order to complement a panel of virus-specific real-time PCR assays with internal controls, a convenient approach is described to generate the several internal controls within single DNA fragment. By applying composite primer technology, PCR primer sequences used in real-time PCR assays were added in 5' and 3' of a stretch of heterologous DNA during consecutive preparative PCRs. The heterologous DNA was used for internal control specific detection by e.g. FRET-hybridisation probes. The presented example of such a multiple internal control DNA contained five internal controls for five competitive LightCycler-PCR assays. All five PCR products derived from the multiple internal control DNA were detected with a single pair of specific FRET-hybridisation probes. The example described proved useful in real-time PCR assays specific for the detection of EBV-, CMV-, VZV- HSV-, and HBV-DNA on the LightCycler instrument. This methodology should enable laboratories to conveniently complement their panel of existing real-time PCR assays with a single multiple internal control DNA.

Base Sequence↗
PubMed2000

Calcium-induced flexibility changes in the troponin C-troponin I complex.

The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.

Animals↗
PubMed2001

The interdomain motions in myosin subfragment 1.

The interdomain motions in myosin subfragment 1 (S1) were studied by steady-state and time-resolved fluorescence of tryptophan residues and N-(iodoacetyl)-N'-(5-sulfo-1-naphtyl)ethylenediamine (AEDANS) attached to Cys178 of alkali light chain 1 (A1) exchanged into S1. The efficiency of fluorescence resonance energy transfer (FRET) from tryptophan residues of motor domain to AEDANS at A1 decreased dramatically after addition of ATP to S1A1-AEDANS. The efficiency of FRET calculated from the crystal structure of chicken S1 corresponded to the experimental one measured in the presence of ATP. The results showed that AEDANS at Cys178 of A1 became more mobile and distant from the motor domain of S1 upon ATP binding. These findings led to the suggestion that a release of the products of ATP hydrolysis and power stroke might be associated with movement of light chain-binding domain towards the N-terminal domain of S1.

Animals↗
PubMed2002

Measuring protein conformational changes by FRET/LRET.

Fluorescence resonance energy transfer (FRET) provides a unique means of measuring interatomic distances in biological molecules in real time. Recent advances have been made in the application of this technique to studies of conformational changes in proteins. New ways of introducing fluorescence probes into proteins, newly developed fluorescence probes, and progress in the technologies for fluorescence signal detection have greatly expanded the range of applications of FRET. In particular, studies of conformational changes in proteins at a single molecule level and in the native in vivo context of a living cell are now possible.

Fluorescence Resonance Energy Transfer↗
PubMed2003

EGF regulation of PITP dynamics is blocked by inhibitors of phospholipase C and of the Ras-MAP kinase pathway.

Phosphatidylinositol transfer proteins (PITP) function in signal transduction and in membrane traffic. Studies aimed at elucidating the mechanism of action of PITP have yielded a singular theme; the activity of PITP stems from its ability to transfer phosphatidylinositol (PI) from its site of synthesis to sites of cellular activity and to stimulate the local synthesis of phosphorylated forms of PI. The participation of various phosphoinositides in EGF signal transduction and in the trafficking of the EGF receptors is well documented. Using fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET) between EGFP-PITP proteins and fluorescently labeled phospholipids, we report that PITPalpha and PITPbeta can dynamically interact with PI or PC at the plasma membrane when stimulated with EGF. Additionally, PITPbeta is localized at the Golgi, and EGF stimulation resulted in enhanced FRET. Inhibitors of the PLC and the Ras/MAP kinase pathway were both able to inhibit the EGF-stimulated interaction of PITPalpha with PI at the plasma membrane. The mobility of PITP proteins was determined by using fluorescence recovery after photobleaching (FRAP), and EGF stimulation reduced the mobility at the plasma membrane. We conclude that the dynamic behavior of PITPalpha and PITPbeta in vivo is a regulated process involving multiple mechanisms.

Animals↗