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PubMed2005

Dependence of the size of the initially collapsed form during the refolding of barstar on denaturant concentration: evidence for a continuous transition.

Two-site fluorescence resonance energy transfer (FRET) measurements have been made to determine how two intra-molecular distances contract in the sub-millisecond collapse reaction that occurs initially during the refolding of the small protein barstar. FRET measurements were made on two, single-Cys and single-Trp-containing mutant forms of barstar, Cys25 and Cys62, in each of which a thionitrobenzoate (TNB) adduct was attached to the cysteine thiol. In each protein, the core tryptophan, Trp53, acted as the FRET donor, and the TNB adduct, located either at C25 or at C62, acted as the FRET acceptor. The stabilities as well as observable folding kinetics of the Cys25 and Cys62 mutant proteins were found to be identical. The presence of the TNB adduct on the cysteine did not alter the stability or folding kinetics of either protein. Thus, the FRET-monitored changes in the two labeled mutant proteins, Cys25-TNB and Cys62-TNB, could be compared directly. Refolding was commenced from unfolded protein in 8M urea, and both the Trp53 to C25-TNB distance and the Trp53 to C62-TNB distance were found to contract upon dilution of urea. The extent of contraction of each distance, which was measured at a few milliseconds of refolding, was dependent continuously on the concentration of urea present during refolding, and was different for the two distances. For either FRET pair, the gradual contraction of distance with a decrease in the concentration of urea in which refolding occurs, was continuous with the contraction of the polypeptide chain that is seen with a decrease in the concentration of urea in the range in which the protein remains completely unfolded. It therefore appears that the products of the initial sub-millisecond refolding reaction of barstar are collapsed forms, whose dimensions do not change cooperatively in an all-or-none manner, but instead, change gradually with a change in concentration of urea. Thus, the sub-millisecond polypeptide chain collapse reaction of barstar upon denaturant dilution, appears to be a continuous structural transition.

Bacterial Proteins↗

PubMed2003

Merging fluorescence resonance energy transfer and expressed protein ligation to analyze protein-protein interactions.

Determination of protein oligomerization state can be technically challenging. We have combined the methods of expressed protein ligation (EPL) and fluorescence resonance energy transfer (FRET) for the analysis of protein homo-oligomerization states. We have attached fluorescein (donor) and rhodamine (acceptor) chromophores via dipeptide linkages to the C-termini of three recombinant proteins and examined the potential for FRET between mixtures of these semisynthetic proteins. The known protein dimer (glutathione S-transferase) showed evidence of FRET and the known protein monomer (SH2 domain phosphatase-1) did not display FRET. Using this method, the previously uncharacterized circadian rhythm enzyme, serotonin N-acetyltransferase, displayed significant FRET, indicating its likely propensity for dimerization or more complex oligomerization. These results establish the potential of the union of EPL and FRET in the analysis of protein-protein interactions and provide insight into the unusual enzymatic behavior of a key circadian rhythm enzyme.

Amino Acid Sequence↗

PubMed2000

Partition of membrane probes in a gel/fluid two-component lipid system: a fluorescence resonance energy transfer study.

A non-ideal lipid binary mixture (dilauroylphosphatidylcholine/distearoylphosphatidylcholine), which exhibits gel/fluid phase coexistence for wide temperature and composition ranges, was studied using photophysical techniques, namely fluorescence anisotropy, lifetime and resonance energy transfer (FRET) measurements. The FRET donor, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanol amine, and a short-tailed FRET acceptor, 1,1'-didodecil-3,3,3',3'-tetramethylindocarbocyanine (DiIC12(3)), were shown to prefer the fluid phase by both intrinsic anisotropy, lifetime and FRET measurements, in agreement with published reports. The other studied FRET acceptor, long-tailed probe 1,1'-dioctadecil-3,3,3',3'-tetramethylindocarbocyanine (DiIC18(3)), is usually reported in the literature as partitioning mainly to the gel. While intrinsic lifetime studies indeed indicated preferential partition of DiIC18(3) into a rigidified environment, FRET analysis pointed to an increased donor-acceptor proximity as a consequence of phase separation. These apparently conflicting results were rationalized on the basis of segregation of DiIC18(3) to the gel/fluid interphase. In order to fluid-located donors sense these interphase-located acceptors, fluid domains should be small (not exceed approximately 10-15 nm). It is concluded that membrane probes which apparently prefer the gel phase may indeed show a non-random distribution in this medium, and tend to locate in an environment which simultaneously leads to less strict packing constraints and to favorable hydrophobic matching interactions.

4-Chloro-7-nitrobenzofurazan↗

PubMed2002

Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy.

Fluorescence resonance energy transfer (FRET) resolved by multifocal multiphoton microscopy (MMM) was successfully used to measure transport phenomena in living cells. We expressed different pairs of CFP-/YFP-fusion proteins involved in retrograde Golgi-to-ER transport to analyze sorting of the occupied KDEL-receptor into retrograde transport vesicles triggered by application of the external cholera toxin mutant CTXK63. FRET observed as a sensitized emission of the acceptor was confirmed by acceptor photobleaching and the dequenching of the donor was measured. FRET-MMM data obtained from single cells were compared with bulk cell experiments employing spectrofluorimetry. The importance of controlling the degree of overexpression of CFP-/YFP-fusion proteins for FRET analysis is stressed in this article. Using MMM we showed for the first time that FRET can be measured across the Golgi membrane. Finally, FRET-MMM records performed continuously over 2 h allowed to analyze intracellular retrograde transport and sorting events and to discuss these mechanisms on a single cell level.

Bacterial Proteins↗

PubMed2001

Intramolecular fluorescence resonance energy transfer system with coumarin donor included in beta-cyclodextrin.

In aqueous solutions, the fluorescence of the intramolecular fluorescence resonance energy-transfer (FRET) system 1 was strongly quenched, because of close contact between the donor and acceptor moieties. FRET occurred, and the acceptor fluorescence was increased, by adding beta-cyclodextrin (beta-CD) to aqueous solutions of 1. Spectral analysis supported the idea that the FRET enhancement was due to the formation of an inclusion complex of the coumarin moiety in beta-CD, resulting in separation of the fluorophores. On the basis of this result, we propose that covalent binding of coumarin to beta-CD will provide a FRET cassette molecule. So, compound 2 bearing beta-CD covalently was designed and synthesized. Fluorescence intensity of 2 was enhanced markedly compared to the intensity of 3. Applying this FRET system, various FRET probes that will be useful for ratio imaging and also the high-throughput screening will be provided.

Chemical Phenomena↗

PubMed2006

Selective determination of native fluorescent bioamines through precolumn derivatization and liquid chromatography using intramolecular fluorescence resonance energy transfer detection.

In this paper, we introduce a novel approach for the highly selective and sensitive analysis of native fluorescent bioamines (indoleamines and catecholamines). This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the bioamines' amino groups. In this detection process, we monitored the FRET from the native fluorescent moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 15 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated the FRET most effectively. The OPA derivatives of the native fluorescent bioamines emitted OPA fluorescence (445 nm) through an intermolecular FRET process when they were excited at the excitation maximum wavelengths of the native fluorescent bioamines (280 nm). The generation of FRET was confirmed through comparison with the analysis of a nonfluorescent amine (isoleucine) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the indoleamines and catecholamines when performing LC on an ODS column. The detection limits (signal-to-noise ratio, 3) for the indoleamines and catecholamines, at a 20-muL injection volume, were 17-120 and 28-200 fmol, respectively. The sensitivity of the intramolecular FRET-forming derivatization method is higher than those of systems that take advantage of both native fluorescence detection (i.e., without derivatization) and the conventional detection of OPA derivatives. Furthermore, this method provides enough selectivity and sensitivity for the determination of the indoleamines present in the urine of healthy humans.

Biogenic Amines↗

PubMed1999

A fluorescent indicator for tyrosine phosphorylation-based insulin signaling pathways.

A fluorescent indicator for tyrosine phosphorylation-based insulin signaling is described. Upon binding of insulin to cell-surface insulin receptor, the receptor phosphorylates tyrosine residues of insulin receptor substrate 1 (IRS-1) in the cell. A fluorescent indicator was designed by using synthetic phosphopeptide pY939 derived from the tyrosine phosphorylation domain of IRS-1 and its target protein SH2N containing an N-terminal SH2 domain of PI 3-kinase. The SH2N protein and pY939 phosphopeptide were labeled with fluorescein (F-SH2N) and tetramethylrhodamine (T-pY939), respectively. Formation of a F-SH2N-T-pY939 complex (termed a fluorescence resonance energy-transfer (FRET) pair) was evaluated from a change in a fluorescence emission spectrum based on FRET between the two fluorophores. The FRET pair was formed to dissociate in competition with the unlabeled pY939 phosphopeptide, resulting in a decrease in a pY939 phosphopeptide-dependent FRET emission at 580 nm and causing an increase in emission at 520 nm. Tyrosine phosphorylation by the partially purified insulin receptor of substrate peptide Y939 was detected with this formed FRET pair, and resulting changes in fluorescence emission spectra were observed for insulin concentration from about 1.0 x 10(-9) to 1.0 x 10(-6) M. These results indicated that the FRET pair served as a competitive fluorescent indicator for tyrosine phosphorylation-based insulin signaling.

Amino Acid Sequence↗

PubMed1994

The colicin E1 insertion-competent state: detection of structural changes using fluorescence resonance energy transfer.

The single cysteine residue (Cys-505) located in the hydrophobic membrane anchor domain in the colicin E1 COOH-terminal channel peptide was labeled with the thiol-specific fluorescent reagent IAEDANS [5-[[[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid]. The labeling stoichiometry was nearly 1:1 [AEDANS: peptide (mol:mol)]. Eleven single Trp mutants of the channel peptide were prepared, and the FRET efficiency for each Trp residue (donor) and the AEDANS chromophore (acceptor), covalently attached to Cys-505, was measured. The FRET efficiencies for the various donor-acceptor pairs ranged from 15% to approximately 100% for the native peptide in solution (pH6.0). The FRET efficiency for the W-507 channel peptide-AEDANS adduct approached 100% since this adduct showed no detectable Trp fluorescence. Activation of the channel peptide to the insertion-competent state upon addition of the nonionic detergent octyl beta-D-glucoside [10,000:1 detergent: peptide (mol:mol)] resulted in decreased FRET efficiencies. The detergent-activated colicin E1 channel peptide-AEDANS adducts possessed significant in vitro channel activity at pH 6.0. The relative changes in the FRET efficiencies upon peptide activation ranged from -1% (W-495 channel peptide-AEDANS adduct) to 48% (W-355 channel peptide-AEDANS adduct). A direct correlation existed between the relative change in FRET efficiency upon channel peptide activation and the position of the Trp (donor) residue within the channel peptide primary sequence (higher relative delta E the closer the Trp donor was to the NH2 terminus), except for the W-484 channel peptide-AEDANS adduct, which showed a higher relative delta E than either W-443 or W-460 channel peptide-AEDANS adducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2006

Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins.

Green fluorescent protein and its variants are frequently used as Förster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (<or=5%). We propose that this disparity primarily arises from the differential response of GFP and YFP on intramolecular association with the donor probe. Studies with isolated GFP and YFP at high concentrations show that they dimerize with similar K(d) values but the spectrum shifts toward the protonated state only with GFP. On excitation at 380 nm, the protonated GFPH emits at 510 nm via excited-state proton transfer, giving the appearance of extensive FRET. These findings have important implications for FRET measurements using GFP-type probes because they give rise to changes in donor and acceptor emission ratios through processes other than FRET and complicate the extraction of the true degree of energy transfer from experimental data. Furthermore, the unknown orientation factor prevents the distance of the lever arm swing from being derived from these FRET changes.

Animals↗

PubMed2006

Analysis of photobleaching in single-molecule multicolor excitation and Förster resonance energy transfer measurements.

Dye photobleaching is a major constraint of fluorescence readout within a range of applications. In this study, we investigated the influence of photobleaching in fluorescence experiments applying multicolor laser as well as Förster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor photobleaching, cross-correlation analysis of single-molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, Tg - Tr, of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET-active subpopulations, reducing bias from FRET-inactive molecules. The observations made are of strong relevance for and demand a careful choice of laser action in multicolor and FRET experiments, in particular when performed at or close to saturation.

Carbocyanines↗

PubMed2006

Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis.

We establish a probability distribution analysis (PDA) method for the analysis of fluorescence resonance energy transfer (FRET) signals to determine with high precision the originating value of a shot-noise-limited signal distribution. PDA theoretical distributions are calculated explicitly including crosstalk, stochastic variations, and background and represent the minimum width that a FRET distribution must have. In this way an unambiguous distinction is made between shot-noise distributions and distributions broadened by heterogeneities. This method simultaneously and effectively extracts highly resolved information from FRET distributions. The theoretical histograms match the exact profile of histograms generated from constant transfer efficiency experimental data with a chi2 near unity. The chi2 surface suggests an ultimate level of precision with FRET of < 1% of the Förster radius. Distributions of FRET signals in donor-acceptor-labeled DNA were unambiguously identified as being broader than shot-noise variations could explain. A model describing a Gaussian distribution of distances was tested with the PDA method and demonstrated 5 A inhomogeneities due to dye motions. The capability of this method to recover quantitative information from FRET distributions has potential applications for studying molecular conformations and dynamics. Potential sources for artifacts such as acceptor photobleaching, spectrally different observation volumes, and fluctuations of the Förster radius are ruled out.

DNA↗

PubMed2006

Spectral Förster resonance energy transfer detection of protein interactions in surface-supported bilayers.

Förster resonance energy transfer (FRET), a fluorescence detection technique, is often used for sensing molecular interactions in solution and in membranes. Here we show that (1) FRET spectra can be recorded in single bilayers, supported on a surface, and (2) the fluorescein/rhodamine dye pair is an adequate reporter of FRET when spectral detection is used. Thus, measurements pertaining to molecular interactions in membranes can be carried out in supported bilayers. Spectral FRET has advantages over imaging FRET, which monitors only signal amplitudes at certain wavelength. There are also advantages to performing spectral FRET measurements in supported bilayers as compared to free liposomes in suspension. However, the spectral properties of dyes can be altered in an unexpected manner in an ordered bilayer structure on a surface, such that fluorescence detection in surface-supported bilayers is not always trivial.

Fluorescence Resonance Energy Transfer↗

PubMed2000

Marking synaptic activity in dendritic spines with a calpain substrate exhibiting fluorescence resonance energy transfer.

Excitatory synaptic activity can evoke transient and substantial elevations of postsynaptic calcium. Downstream effects of elevated calcium include the activation of the calcium-dependent protease calpain. We have developed a reagent that identifies dendritic spines in which calpain has been activated. A fusion protein was expressed that contained enhanced yellow and enhanced cyan fluorescent protein (EYFP and ECFP, respectively) linked by a peptide that included the micro-calpain cleavage site from alpha-spectrin. A PDZ-binding site fused to ECFP anchored this protein to postsynaptic densities. The fusion protein exhibited fluorescence resonance energy transfer (FRET), and diminution of FRET by proteolysis was used to localize calpain activity in situ by fluorescence microscopy. Incubation of the fusion protein with calpain in the presence of calcium resulted in the separation of EYFP and ECFP into monomeric fluorophores. In transiently transfected cell lines and dissociated hippocampal neurons, FRET was diminished by raising intracellular calcium levels with an ionophore or with glutamatergic agonists. Calpain inhibitors blocked these changes. Under control conditions, FRET levels in different dendritic spines of cultured neurons and in hippocampal slices were heterogeneous but showed robust decreases upon treatment with glutamatergic agonists. Immunostaining of cultured neurons with antibodies to a spectrin epitope produced by calpain-mediated digestion revealed an inverse correlation between the amount of FRET present at postsynaptic elements and the concentration of spectrin breakdown products. These results suggest that the FRET methodology identifies sites of synaptically induced calpain activity and that it may be useful in analyzing synapses undergoing changes in efficacy.

Amino Acid Sequence↗

PubMed2001

Galpha i3 binding to calnuc on Golgi membranes in living cells monitored by fluorescence resonance energy transfer of green fluorescent protein fusion proteins.

Galphai3 is found both on the plasma membrane and on Golgi membranes. Calnuc, an EF hand protein, binds both Galphai3 and Ca(2+) and is found both in the Golgi lumen and in the cytoplasm. To investigate whether Galphai3 binds calnuc in living cells and where this interaction takes place we performed fluorescence resonance energy transfer (FRET) analysis between Galphai3 and calnuc in COS-7 cells expressing Galphai3-yellow fluorescent protein (YFP) and calnuc-cyan fluorescent protein (CFP). The tagged proteins have the same localization as the endogenous, nontagged proteins. When Galphai3-YFP and calnuc-CFP are coexpressed, a FRET signal is detected in the Golgi region, but no FRET signal is detected on the plasma membrane. FRET is also seen within the Golgi region when Galphai3 is coexpressed with cytosolic calnuc(DeltaN2-25)-CFP lacking its signal sequence. No FRET signal is detected when Galphai3(DeltaC12)-YFP lacking the calnuc-binding region is coexpressed with calnuc-CFP or when Galphai3-YFP and calnuc(DeltaEF-1,2)-CFP, which is unable to bind Galphai3, are coexpressed. Galphai3(G2AC3A)-YFP lacking its lipid anchors is localized in the cytoplasm, and no FRET signal is detected when it is coexpressed with wild-type calnuc-CFP. These results indicate that cytosolic calnuc binds to Galphai3 on Golgi membranes in living cells and that Galphai3 must be anchored to the cytosolic surface of Golgi membranes via lipid anchors for the interaction to occur. Calnuc has the properties of a Ca(2+) sensor protein capable of binding to and potentially regulating interactions of Galphai3 on Golgi membranes.

Animals↗

PubMed1993

Observing the helical geometry of double-stranded DNA in solution by fluorescence resonance energy transfer.

The efficiency of fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine covalently attached to both 5' termini of a series of double-stranded DNA species (ranging from 8 to 20 bp) was measured. FRET efficiency varied with a dependence compatible with dye-to-dye distances (R) calculated on the basis of double-stranded B-DNA structure; the helical geometry of double-stranded DNA in solution is clearly evident. The experimental data were consistent with a 1/[1 + (R/R0)6] dependence of FRET efficiency characteristic for the Förster dipole-dipole mechanism. The thermal dissociation of the strands of the duplex DNA species can be followed by using FRET, and from these data we have been able to obtain enthalpies of duplex formation in good agreement with earlier measurements using alternative techniques. FRET measurements at very different salt concentrations can be accurately compared. We conclude that FRET is a reliable and valuable method for studying structure and conformational transitions in nucleic acids.

Base Sequence↗

PubMed1995

Fluorescence resonance energy transfer reveals interleukin (IL)-1-dependent aggregation of IL-1 type I receptors that correlates with receptor activation.

Fluorescence resonance energy transfer (FRET) was used to investigate whether interleukin-1 (IL-1) causes the aggregation of IL-1 type I receptors (IL-1 RI) at the cell surface. For these experiments, a noncompetitive anti-IL1 RI monoclonal antibody, M5, was labeled separately with a donor probe, fluorescein isothiocyanate, or with an acceptor carbocyanine probe, Cy3. Donor-labeled M5 and acceptor-labeled M5 were simultaneously bound to transfected mouse IL-1 RI on either C-127 mouse mammary carcinoma cells or on Chinese hamster ovary (CHO)-K1 cells, and the ratio of acceptor emission at 590 nm to donor emission at 525 nm (excitation at 488 and 514 nm) was monitored with flow cytometry as an indicator of FRET. Addition of a saturating concentration of human IL-1 alpha at 22 degrees C causes a time-dependent increase in FRET for both cell lines that indicates IL-1-dependent self-association of IL-1 RI. Binding of the IL-1 receptor antagonist at 22 degrees C causes little or no FRET for both cell lines, indicating a correlation between receptor aggregation and the ability of the ligand to stimulate a functional response. When donor-labeled and acceptor-labeled Fab fragments of M5 are used to monitor FRET, IL-1 alpha causes efficient energy transfer in the CHO-K1 cells at 22 degrees C, but not at 4 degrees C. In contrast, IL-1 alpha causes much less FRET at 22 degrees C in C-127 cells when the M5 Fab fragments are used instead of the intact bivalent M5. In a striking parallel, IL-1 alpha-dependent activation of prostaglandin E2 production depends on the bivalent M5 antibody in the C-127 cells, but is independent of this monoclonal antibody in the CHO-K1 cells. These results provide a strong correlation between the ability of IL-1 to cause the aggregation of IL-1 RI and the stimulation of a functional response.

Animals↗

PubMed1997

Soluble amyloid Abeta-(1-40) exists as a stable dimer at low concentrations.

Recent studies have implicated the amyloid Abeta peptide and its ability to self-assemble as key factors in the pathogenesis of Alzheimer's disease. Relatively little is known about the structure of soluble Abeta or its oligomeric state, and the existing data are often contradictory. In this study, we used intrinsic fluorescence of wild type Abeta-(1-40), fluorescence resonance energy transfer (FRET), and gel filtration chromatography to examine the structure of Abeta-(1-40) in solution. We synthesized a series of mono-substituted fluorescent Abeta-(1-40) derivatives to use as donors and acceptors in FRET experiments. We selected fluorescent peptides that exhibit aggregation properties comparable to wild type Abeta for analysis in donor-acceptor pairs; two labeled with 5-(2-((iodoacetyl)amino)ethyl)aminonaphthylene-1-sulfonic acid at Cys-25 or Cys-34 and fluorescein maleimide at Cys-4 or Cys-7. Another peptide containing a Trp substitution at position 10 was used as an acceptor for the intrinsic Tyr fluorescence of wild type Abeta-(1-40). Equilibrium studies of the denaturation of Abeta-(1-40) by increasing concentrations of dimethyl sulfoxide (Me2SO) were conducted by monitoring fluorescence, with a midpoint value for the unfolding transition of both the substituted and wild type peptides at among 40 and 50% Me2SO. Abeta-(1-40) is well solvated and largely monomeric in Me2SO as evidenced by a lack of FRET. When donor and acceptor Abeta derivatives are mixed together in Me2SO and then diluted 10-fold into aqueous Tris-HCl buffer at pH 7.4, efficient FRET is observed immediately for all pairs of fluorescent peptides, indicating that donor-acceptor dimers exist in solution. FRET is abolished by the addition of an excess of unlabeled Abeta-(1-40), demonstrating that the fluorescent peptides interact with wild type Abeta-(1-40) to form heterodimers that do not exhibit FRET. The Abeta-(1-40) dimers appear to be very stable, because no subunit exchange is observed after 24 h between fluorescent homodimers. Gel filtration confirms that nanomolar concentrations of 14C-labeled Abeta-(1-40) and fluorescein-labeled Abeta-(1-40) elute at the same dimeric position as wild type Abeta-(1-40), suggesting that soluble Abeta-(1-40) is also dimeric at more physiologically plausible concentrations.

Amyloid beta-Peptides↗

PubMed2000

Stable SNARE complex prior to evoked synaptic vesicle fusion revealed by fluorescence resonance energy transfer.

Although it is clear that soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex plays an essential role in synaptic vesicle fusion, the dynamics of SNARE assembly during vesicle fusion remain to be determined. In this report, we employ fluorescence resonance energy transfer technique to study the formation of SNARE complexes. Donor/acceptor pair variants of green fluorescent protein (GFP), cyan fluorescent protein (CFP), and yellow fluorescent protein (YFP) are fused with the N termini of SNAP-25 and synaptobrevin, respectively. In vitro assembly of SNARE core complex in the presence of syntaxin shows strong fluorescence resonance energy transfer (FRET) between the CFP-SNAP-25 and YFP-synaptobrevin. Under the same conditions, CFP fused to the C terminus of SNAP-25, and YFP- synaptobrevin have no FRET. Adenovirus-mediated gene transfer is used to express the fusion proteins in PC12 cells and cultured rat cerebellar granule cells. Strong FRET is associated with neurite membranes and vesicular structures in PC12 cells co-expressing CFP-SNAP-25 and YFP-synaptobrevin. In cultured rat cerebellar granule cells, FRET between CFP-SNAP-25 and YFP-synaptobrevin is mostly associated with sites presumed to be synaptic junctions. Neurosecretion in PC12 cells initiated by KCl depolarization leads to an increase in the extent of FRET. These results demonstrate that significant amounts of stable SNARE complex exist prior to evoked synaptic vesicle fusion and that the assembly of SNARE complex occurs during vesicle docking/priming stage. Moreover, it demonstrates that FRET can be used as an effective tool for investigating dynamic SNARE interactions during synaptic vesicle fusion.

Animals↗