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PubMed2004

Applying spectral fingerprinting to the analysis of FRET images.

Förster resonance energy transfer (FRET) allows one to study interactions between two fluorescently labeled molecules (donors and acceptors) at distances on the order of 5 nm. Many studies have described methods of how to measure the efficiency of FRET. However, few have addressed the question of how fluorescence from unpaired donors and acceptors can be determined in addition to that from FRET-pairs and how the signal-to-noise ratio (SNR) of such estimates depends on the presence of the partner species. Such knowledge, however, is essential for many biological applications, in which-after initial characterization of the spectral properties of a well-defined donor-acceptor complex-the in vivo affinity and stoichiometry of the complex is of interest. Here, we provide a theoretical analysis on how spectral fingerprinting can be applied to separate fluorescence of FRET pairs from that originating from unpaired donors and acceptors and how to select imaging parameters to optimize the SNR of the estimates. Thereby, we uncover a fundamental problem in this application and discuss ways to evade its adverse consequences. We compare the expected resolution of traditional FRET measures with that of optimized spectral fingerprinting and analyze the resolution of a method for FRET measurements that combines spectral with fluorescence lifetime information.

Cells↗
PubMed1993

Reduction of fretting corrosion of Ti-6Al-4V by various surface treatments.

Titanium and titanium-6% aluminum-4% vanadium (Ti-6Al-4V) are known to be biocompatible and corrosion resistant. However, there have been numerous reports of elevated tissue levels of titanium due to passive dissolution, wear, or fretting corrosion of implants. Studies were undertaken to determine whether the fretting corrosion of Ti-6Al-4V could be reduced by surface treatment of one or both surfaces in a fretting situation. Three different surface treatments were studied: ion implantation, physical vapor deposition nitriding, and plasma ion nitriding. The specimens used were screws fretting against the countersinks of a two-hole plate. Fretting corrosion was assessed by weight loss, by chemical analysis of test solutions, and by scanning electron microscopy. Surface treatment of one component, the screws, resulted in reduction in the release of titanium to only 18-32% of that seen with the untreated controls. Weight loss of the untreated plates fretted against physical vapor deposition nitrided screws and plasma ion nitrided screws was reduced to 31 and 38% of the control, respectively. The weight loss of plasma nitrided screws was only 30% that of the control. Nitriding of both plates and screws resulted in a further decrease in plate weight loss and metal release. Plasma ion nitriding of both components had the most significant effect, with the weight loss and titanium release being only 11 and 2% of the control values, respectively.

Alloys↗
PubMed2004

Conformation of prion protein repeat peptides probed by FRET measurements and molecular dynamics simulations.

We report the combined use of steady-state fluorescence resonance energy transfer (FRET) experiments and molecular dynamics (MD) simulations to investigate conformational distributions of the prion protein (PrP) repeat system. FRET was used for the first time to probe the distance, as a function of temperature and pH, between a donor Trp residue and an acceptor dansyl group attached to the N-terminus in seven model peptides containing one to three repeats of the second decarepeat of PrP from marsupial possum (PHPGGSNWGQ)nG, and one and two human PrP consensus octarepeats (PHGGGWGQ)nG. In multirepeat peptides, single-Trp mutants were made by replacing other Trp(s) with Phe. As previous work has shown PrP repeats do not adopt a single preferred stable conformation, the FRET values are averages reflecting heterogeneity in the donor-acceptor distances. The T-dependence of the conformational distributions, and derived average dansyl-Trp distances, were obtained directly from MD simulation of the marsupial dansyl-PHPGGSNWGQG peptide. The results show excellent agreement between the FRET and MD T-dependent distances, and demonstrate the remarkable sensitivity and reproducibility of the FRET method in this first-time use for a set of disordered peptides. Based on the results, we propose a model involving cation-pi or pi-pi His-Trp interactions to explain the T- (5-85 degrees C) and pH- (6.0, 7.2) dependencies on distance, with HW i, i + 4 or WH i, i + 4 separations in sequence being more stable than HW i, i + 6 or WH i, i + 6 separations. The model has peptides adopting loosely folded conformations, with dansyl-Trp distances very much less than estimates for fully extended conformations, for example, approximately 16 vs. 33, approximately 21 vs. 69, and approximately 22 vs. 106 A for 1-3 decarepeats, and approximately 14 vs. 25 and approximately 19 vs. 54 A for 1-2 octarepeats, respectively. The study demonstrates the usefulness of combining FRET with MD, a combination reported only once previously. Initial "mapping" of the conformational distribution of flexible peptides by simulation can assist in designing and interpreting experiments using steady-state intensity methods, and indicating how time-resolved or anisotropy methods might be used.

Amino Acid Sequence↗
PubMed2004

Differences in the fretting corrosion of metal-metal and ceramic-metal modular junctions of total hip replacements.

The use of modular interlocking components is a central design feature of total joint replacements. In this investigation we hypothesized that clinically available ceramic-metal modular connections used in total hip arthroplasty release more metal through fretting corrosion than traditional metal-metal modular connections. This was investigated using an in vitro comparison of ceramic (zirconia, ZrO2) and metal (Co-alloy) femoral-head fretting upon Co-alloy stem components. In vitro fretting corrosion testing consisted of potentiodynamic monitoring and analysis of metal release from zirconia and Co-alloy 28 mm femoral heads with similar surface roughnesses (Ra=0.46 microm) on identical Co-alloy stems at 2.2 kN for 1x10(6) cycles at 2 Hz. In contrast to our original hypothesis, we found greater metal release (approximately 11-fold increase in Co and 3-fold increase in Cr) and potentiodynamic fretting of metal-metal modular junctions when compared to ceramic-metal. Potentiodynamic testing demonstrated that lower initial voltages (-266<153 mV), greater maximum voltage changes (116>56 mV, p<0.05, t-test) and voltage variability (3>0.5 mV, p<0.05, t-test) were associated with the open circuit potentials of Co-alloy on Co-alloy junctions when compared to zirconia on Co-alloy junctions. In this study of a single total hip replacement stem and head design, zirconia heads mated with Co-alloy stems produced less fretting than Co-alloy heads mated with Co-alloy stems. Although further studies are necessary with a variety of implant designs and under different experimental conditions, the evidence presented here should, in part, alleviate concerns of increases in fretting corrosion at modular junctions of ceramic-metal coupled components.

Adult↗
PubMed2005

Fluorescence resonance energy transfer (FRET) microscopy in living cells as a novel tool for the study of cytokine action.

Fluorescence resonance energy transfer (FRET) microscopy was used to study interactions between proteins in intact cells. We showed that growth hormone (GH) causes transient homodimerization of GH receptors tagged with yellow or cyan fluorescent proteins. The peak of FRET signaling occurred 2 to 4 min after hormonal stimulation and was followed by a decrease in FRET signal. Repeating those experiments in cells pretreated with the inhibitor of internalization methyl-beta-cyclodextrin, or in potassium-depleted cells showed no difference in the kinetics of FRET signaling as compared with the non-treated cells, indicating that the decrease in FRET signal does not result from receptor internalization by the pathways inhibited by methyl-beta-cyclodextrin or potassium depleted but might occur by other pathways of internalization. Using a similar methodology, we also demonstrated that ovine placental lactogen (oPL) causes transient heterodimerization of GH and prolactin (PRL) receptors 2.5 to 3 min after oPL application. On the other hand, oGH or oPRL had no effect at all, further substantiating the finding the oPL, which lacks a specific receptor, acts in homologous systems by heterodimerization of GH and PRL receptors. We also demonstrated that both PRL and leptin (LEP) are capable of transactivation of the oncogenic receptors erbB2 and erbB3. Upon PRL or LEP stimulation of HEK-293T cells transfected with LEP or PRL receptors and erbB2 or erbB3, erbB proteins are first phosphorylated and then activate MAPK (erk1/erk2). However, the FRET experiments failed to document any evidence of a direct interaction between erbB2 and the PRL or LEP receptors, suggesting that erbB activation probably occurs via activated JAK2, translocated from the respective receptors to erbB2.

Animals↗
PubMed2002

FRET study of a trifluorophore-labeled DNAzyme.

A fluorescence resonance energy transfer (FRET) study of biomolecules typically employs two fluorophores. The increasing number of branches and complexity of biomolecules call for simultaneously monitoring structures and dynamics of several branches in a single system. Furthermore, despite recent studies that show DNAzymes can be a stable and cost-effective alternative to protein and ribozymes for pharmaceutical and biotechnological applications, no FRET study of DNAzymes has been reported. Here, we describe the FRET study of a trifluorophore-labeled "8-17" DNAzyme, in which each of the three branches is labeled with a different fluorophore. From the study, we found that the (ratio)(A) method that has been commonly used in dual-fluorophore-labeled systems is also applicable to trifluorophore-labeled systems. However, while both FRET efficiency and fluorophore-to-fluorophore distance can be used to measure FRET in dual-fluorophore-labeled systems, only the average distance should be used in trifluorophore-labeled systems. The ability to monitor all three branches in a single system allowed us to reveal new metal-ion-dependent conformational changes in the DNAzyme. The trifluorophore-labeled "8-17" DNAzyme has been found to adopt a two-step folding process in the presence of Zn(2+). Each step is induced by one Zn(2+) binding, with apparent dissociation constants of 19 microM and 260 microM for binding the first and second Zn(2+), respectively. The trifluorophore FRET results are verified by a dual-labeled control experiment. The results demonstrated that the trifluorophore-labeled system is simple and yet powerful in studying complicated biomolecular structure and dynamics and is capable of revealing new sophisticated structural changes that may have functional implications.

DNA, Catalytic↗
PubMed2003

Two-step FRET as a structural tool.

The power of FRET to study molecular complexes is expanded by the use of two or more donor/acceptor pairs. A general theoretical framework for distance measurements in three-chromophore systems is presented. Three energy transfer schemes applicable to many diverse situations are considered: (I) two-step FRET relay with FRET between the first and second chromophores and between the second and third, (II) FRET from a single donor to two different acceptors, and (III) two-step FRET relay with FRET also between the first and third chromophores. Equations for the efficiencies involving multiple energy transfer steps are derived for both donor quenching and sensitized emission measurements. The theory is supported by experimental data on model systems of known structure using steady-state donor quenching, lifetime quenching, and sensitized emission. The distances measured in the three-chromophore systems agree with those in two-chromophore systems and molecular models. Finally, labeling requirements for diagnosis of the energy transfer scheme and subsequent distance measurements are discussed.

DNA↗
PubMed2005

Quantitative FRET imaging of leptin receptor oligomerization kinetics in single cells.

BACKGROUND INFORMATION: Leptin, an adipocyte-secreted hormone, signals through activation of its membrane-embedded receptor (LEPR). To study the leptin-induced events occurring in short (LEPRa) and long (LEPRb) LEPRs in the cell membrane, by FRET (fluorescence resonance energy transfer) methodology, the respective receptors, tagged at their C-terminal with CFP (cyan fluorescent protein) or YFP (yellow fluorescent protein), were prepared. RESULTS: The constructs encoding mLEPRa (mouse LEPRa)-YFP and mLEPRa-CFP, mLEPRb-YFP and mLEPRb-CFP were tested for biological activity in transiently transfected CHO cells (Chinese-hamster ovary cells) and HEK-293T cells (human embryonic kidney 293 T cells) for activation of STAT3 (signal transduction and activators of transcription 3)-mediated LUC (luciferase) activity and binding of radiolabelled leptin. All four constructs were biologically active and were as potent as their untagged counterparts. The localization pattern of the fused protein appeared to be confined almost entirely to the cell membrane. The leptin-dependent interaction between various types of receptors in fixed cells were studied by measuring FRET, using fluorescence lifetime imaging microscopy and acceptor photobleaching methods. CONCLUSIONS: Both methods yielded similar results, indicating that (1) leptin receptors expressed in the cell membrane exist mostly as preformed LEPRa/LEPRa or LEPRb/LEPRb homo-oligomers but not as LEPRb/LEPRa hetero-oligomers; (2) the appearance of transient leptin-induced FRET in cells transfected with LEPRb/LEPRb reflects both a conformational change that leads to closer interaction in the cytosolic part and a higher FRET signal, as well as de novo homo-oligomerization; (3) in LEPRa/LEPRa, exposure to leptin does not lead to any increase in FRET signalling as the proximity of CFP and YFP fluorophores in space already gives maximal FRET efficiency of the preoligomerized receptors.

Animals↗
PubMed2005

Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation.

Fluorescence resonance energy transfer (FRET) between a donor (D) and an acceptor (A) at the single-molecule level currently provides qualitative information about distance, and quantitative information about kinetics of distance changes. Here, we used the sorting ability of confocal microscopy equipped with alternating-laser excitation (ALEX) to measure accurate FRET efficiencies and distances from single molecules, using corrections that account for cross-talk terms that contaminate the FRET-induced signal, and for differences in the detection efficiency and quantum yield of the probes. ALEX yields accurate FRET independent of instrumental factors, such as excitation intensity or detector alignment. Using DNA fragments, we showed that ALEX-based distances agree well with predictions from a cylindrical model of DNA; ALEX-based distances fit better to theory than distances obtained at the ensemble level. Distance measurements within transcription complexes agreed well with ensemble-FRET measurements, and with structural models based on ensemble-FRET and x-ray crystallography. ALEX can benefit structural analysis of biomolecules, especially when such molecules are inaccessible to conventional structural methods due to heterogeneity or transient nature.

Biophysics↗
PubMed2005

Comparison of different fluorescence fluctuation methods for their use in FRET assays: monitoring a protease reaction.

We compare the accuracy of a variety of Fluorescence Fluctuation Spectroscopy (FFS) methods for the study of Förster Resonance Energy Transfer (FRET) assays. As an example, the cleavage of a doubly labeled, FRET-active peptide substrate by the protease Trypsin is monitored and analyzed using methods based on fluorescence intensity, Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA). The presented fluorescence data are compared to High-Pressure Liquid Chromatography (HPLC) data obtained from the same assay. The HPLC analysis discloses general disadvantages of the FRET approach, such as incomplete labeling and the need for aliquots. However, the simultaneous use of two photon detectors monitoring the fluorescence signal of both labels significantly improves the analysis. In particular, the two global analysis tools Two-Dimensional Fluorescence Intensity Distribution Analysis (2D-FIDA) and Two-Color Global Fluorescence Correlation Spectroscopy (2CG-FCS) highlight the potential of a combination of FFS and FRET. While conventional FIDA and FCS auto- or cross-correlation analysis leaves the user with drawbacks inherent in two-color and FRET applications, these effects are overcome by the global analysis on the molecular level. Furthermore, it is advantageous to analyze the unnormalized as opposed to the normalized correlation data when combining any fluorescence correlation method with FRET, since the analysis of the unnormalized data introduces more accuracy and is less sensitive to the experimental drawbacks.

Algorithms↗
PubMed1997

Structural analysis of nucleic acids by using fluorescence resonance energy transfer (FRET).

We examined changes in the extent of fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In our system, FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. While, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. It should help us to estimate the suitability of designed functional molecules, such as antisense DNA and RNA and ribozymes, that target to specific sites.

DNA↗
PubMed1996

Effects of Ca and H2O2 added to RPMI on the fretting corrosion of Ti6Al4V.

Titanium and its alloys have demonstrated considerable success in various surgical procedures including orthopedic, dental, and cardiovascular surgery. However, particulate debris from corrosion and wear is present in a considerable quantity in tissue local to the implant. This study evaluated the effect of Ca, since it is present in both serum and bone, and H2O2, since it is produced through local inflammation, on the amount of titanium release. Four sets of Ti6Al4V plates and Ti6Al4V screws were used. Each set was designated to one of four solutions: RPMI (cell culture growth media), RPMI with CaCl2, RPMI with CaCO3, and RPMI with H2O2. A fretter was used to cause corrosion by creating micromotion between two screws and a two-hole plate of Ti6Al4V. After fretting for 72 h, weight loss of the plate and screws and the amount of Ti and vanadium (V) in solution was used to assess the amount of fretting corrosion which had occurred. Results of weight loss and Ti in solution indicated that the presence of H2O2 increased the amount of particulate debris produced in RPMI as compared with RPMI alone. The addition of CaCl2 to RPMI also increased both weight loss and Ti in solution compared with RPMI alone. The addition of CaCO2, however, did not give values significantly different from RPMI alone. Comparison of weight loss and Ti in solution indicated that the increase in fretting corrosion was not different between RPMI with CaCl2 and RPMI with H2O2. The particulate wear debris from the four solutions was black in color and the size of the particulate produced was compared using a Coulter Multisizer. The results indicated that particles produced in the four solutions were not different, with mean values between 1.324 and 1.100 microns, and they were similar in size to the particulate found in tissues surrounding failed total hip replacements. In order to better understand the role of Ca in the fretting corrosion of Ti6Al4V, energy dispersive x-ray analysis (EDXA) using SEM was used to determine elemental composition of one countersink surface of a plate which had been run four times in RPMI with CaCl2. The presence of Ca in the bulk was not significant (% composition < 0.5%). However, Ca was present in two surface particles which were examined at a magnification of 55,000, with a Ca% composition of 63.2% and 19.2%. While results from this study indicate that both soluble Ca(CaCl2) and H2O2 increase the fretting corrosion of Ti6Al4V, the insoluble form of Ca, which would be found in bone and hydroxyapatite, has no effect. These data indicate that it is important to specify the media used in corrosion, dissolution, and elution experiments.

Alloys↗
PubMed2006

Measurement of two caspase activities simultaneously in living cells by a novel dual FRET fluorescent indicator probe.

BACKGROUND: A number of fluorescent caspase substrates and FRET-based indicators have been developed to study the in vivo activation of caspases, a conserved family of proteases critical in inflammatory, and apoptosis signaling pathways. To date, all substrates have measured only one caspase activity. Here, we describe a FRET-based probe for simultaneously measuring two distinct caspase activities in living cells. METHODS: This probe consists of a CFP-YFP-mRFP fusion protein containing a caspase-3-cleavage motif, DEVD, between CFP and YFP, and a caspase-6-cleavage site, VEID, between YFP and mRFP. DEVDase and VEIDase activities could be assessed simultaneously by monitoring diminished FRET mediated by cleavage of either or both of these protease cleavage sites using flow cytometry. RESULTS: DEVDase and VEIDase activities were completely inhibited by the pan-caspase inhibitor z-VAD-fmk and enhanced by DNA-damaging drugs or by anti-Fas stimulation. DEVD and VEID cleavage specificities were validated by using caspase-3-deficient MCF7-Fas cells and a caspase-6-specific inhibitor. Kinetic analysis with the FRET probe revealed that caspase-3 activation consistently preceded caspase-6 by approximately 30 min following induction of apoptosis. CONCLUSIONS: We have developed a novel FRET-based probe for simultaneous detection of two caspase activities in living cells using flow cytometry. Simultaneous detection of two caspase activities using this probe has clearly provided information of the ordering of caspase-3 and -6 in the apoptotic pathway.

Apoptosis↗
PubMed2006

Issues in confocal microscopy for quantitative FRET analysis.

Previously, we have carried out extensive quantitative analysis of Förster (or fluorescence) resonance energy transfer (FRET) data to show that polymeric IgA receptors and their ligands cluster in endocytic membranes in the process of sorting and trafficking in polarized cells. Here, we use a similar technique to assay the organization and distribution of another membrane-bound receptor: transferrin receptor (TFR) and its ligand, holo-transferrin (Tfn), while explaining the step-by-step measures to be taken for successful quantitative analysis of the FRET data. In particular, methodological issues in FRET quantitative imaging, such as spectral bleed-through and background correction, optimal selection of regions of interest, how to deal with outliers and pooling data and statistical analysis of FRET data, are addressed. Our results indicating a clustered organization of TFR-Tfn complexes fit the well-known homodimeric structure of TFR. These quantitative approaches can be adapted for other biological applications of FRET.

Algorithms↗
PubMed1988

In vitro studies of fretting corrosion of orthopaedic materials.

Two models were used to study fretting corrosion of surgical alloys. In the first, the amount of load and motion between plates and screws was controlled, and corrosion rates of stainless steel and MP35N were determined by measurements of component weight loss and metal ion concentration in the test solutions. Stainless steel had the higher rate of fretting corrosion. The addition of 10% serum to saline resulted in a significant reduction of the corrosion rate of stainless steel and MP35N. The use of stainless steel screws in a mixed-metal combination with MP35N and titanium plates showed minimal effects on the performance of the stainless steel screws but an increase in fretting corrosion of the MP35N plates. In the second model, plates were applied to glass fiber-reinforced plastic tubes as bone analogues and subjected to cyclic axial loads. These results demonstrated a reduction in fretting corrosion rates associated with an increase in screw torque and a decrease in axial load. Corrosion rates were minimal with intact tubes simulating healed fractures and greatest with an oblique-cut fracture simulation. These models have proven useful for the evaluation of fretting corrosion rates of different alloys and for evaluation of variables such as fracture stability.

Alloys↗
PubMed1990

Fretting corrosion studies of universal femoral head prostheses and cone taper spigots.

Short-duration, cyclically loaded, axial, fretting corrosion tests were designed and performed to compare the fretting behaviour of different metal Howmedica universal heads connected to coated and uncoated metal cone taper spigots. Concurrent axial push-on and pull-off taper friction tests were also performed. There was no measurable fretting debris found in any test and SEM studies of the contact surfaces showed no evidence of fretting. It is concluded that no significant, long-term fretting corrosion of the Vitalium Co-Cr-Mo heads on the Vitallium or uncoated Ti-6AI-4V spigots, nor of the Orthinox stainless steel heads on the Orthinox spigots, is likely to occur in vivo.

Corrosion↗
PubMed2005

CaMK-II oligomerization potential determined using CFP/YFP FRET.

Members of the Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) family are encoded throughout the animal kingdom by up to four genes (alpha, beta, gamma, and delta). Over three dozen known CaMK-II splice variants assemble into approximately 12-subunit oligomers with catalytic domains facing out from a central core. In this study, the catalytic domain of alpha, beta, and delta CaMK-IIs was replaced with cyan (CFP) or yellow fluorescent protein (YFP) for fluorescence resonance energy transfer (FRET) studies. FRET, when normalized to total CFP and YFP, reproducibly yielded values which reflected oligomerization preference, inter-subunit spacing, and localization. FRET occurred when individual CFP and YFP-linked CaMK-IIs were co-expressed, but not when they were expressed separately and then mixed. All hetero-oligomers exhibited FRET values that were averages of their homo-oligomeric parents, indicating no oligomeric preference or restriction. FRET for CaMK-II homo-oligomers was inversely proportional to the variable region length. FPs were monomerized (Leu221 to Lys221) for this study, thus eliminating any potential artifact caused by FP-CaMK-II aggregates. Our results indicate that alpha, beta, and delta CaMK-IIs can freely hetero-oligomerize and that increased variable region lengths place amino termini further apart, potentially influencing the rate of inter-subunit autophosphorylation.

Amino Acid Sequence↗
PubMed2006

GFP-based FRET analysis in live cells.

Fluorescence resonance energy transfer (FRET) is a widely utilized optical technique for measuring small distances of 1-10 nm in live cells. In recent years, its application has been greatly popularized by the discovery of green fluorescent protein (GFP) and many improved variants which make good donor-acceptor fluorophore pairs. GFP-based proteins are structurally stable, relatively inert, and can be reliably attached to points of interest. The combination of easy access to the GFP-based FRET technique and its obvious usefulness in many applications can lead to complacency. Potential problems such as light contaminants, e.g., bleed-through and cross-talk, and inconsistent donor and acceptor concentrations are easily overlooked and can lead to errors in FRET calculation and data interpretation. In this article, we outline possible pitfalls of GFP-based FRET and approaches that address these issues, including a "Spectra FRET" technique that can be easily applied to live cell studies.

Animals↗