Use of Forster's resonance energy transfer microscopy to study lipid rafts

Rafts in cell membranes have been a subject of much debate and many models have been proposed for their existence and functional significance. Recent studies using Forster's resonance energy transfer (FRET) microscopy have provided one of the first glimpses into the organization of putative raft components in living cell membranes. Here we discuss how and why FRET microscopy provides an appropriate non-invasive methodology to examine organization of raft components in cell membranes; a combination of homo and hetero-FRET microscopy in conjunction with detailed theoretical analyses are necessary for characterizing structures at nanometre scales. Implications of the physical characteristics of the organization of GPI-anchored proteins in cell membranes suggest new models of lipid-based assemblies in cell membranes based on active principles.     

New donor-acceptor pair for fluorescent immunoassays by energy transfer

A novel F?rster donor-acceptor dye pair for an immunoassay based on resonance energy transfer (RET) is characterized with respect to its photophysical properties. As donor and acceptor, we chose the long-wavelength excitable cyanine dyes Cy5 and Cy5.5, respectively. Due to the perfect spectral overlap, an exceptionally high R(0) value of 68.7 A is obtained in solution. For biochemical applications, antibodies (IgG) are labeled with Cy5, while a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to proteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R(0) value of 85.2 A. At higher dye/protein ratios, the formation of nonfluorescent dimeric species causes a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The F?rster distances could be calculated using the dimer absorption spectra to 83.9 and 83.6 A for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled IgG to the tracer, efficient quenching of Cy5 fluorescence is observed. Steady-state and time-resolved measurements reveal that approximately 50% of the quenching results in F?rster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pair is demonstrated in a homogeneous competitive immunoassay for the pesticide simazine.     

On the revised concept of linear energy transfer

The quantities linear energy transfer or restricted linear energy transfer are utilized in calculations that link absorbed dose to the fluence distribution of a radiation field. The computations provide approximations to absorbed dose in terms of the intermediate quantity cema or reduced cema. With the definition of the restricted linear energy transfer,L , given in ICRU Report 33, the approximation remains imperfect. This study deals with the resulting need for a modified definition ofL , as proposed in a draft report of ICRU. Essential differences between the old and the new definitions are demonstrated. The changed definition permits a rigorous formulation of the dependence between fluence and absorbed dose.     

The evaluation of the age-related characteristics of the structural status of the plasma membrane lipid phase in rat fatty tissue by using the method of inductive-resonance energy transfer

Using the method of inductance-resonance energy transfer from tryptophanyl residues to fluorescent pyrene probe the structural state of plasmatic membranes from adipose tissue of different age rats has been studied. The structural heterogeneity of membrane lipid phase has been revealed. The differences in physical properties of annular and bilayer lipids don't depend on age. During aging the membrane lipid viscosity including lipids of near protein area decreases, the conformation of membrane protein components alters during aging as well. The data on various effectiveness of energy transfer from tryptophanyls to pyrene probe in young and aged animals with stable polypeptide composition of membrane proteins indicates that. The structure of membrane lipid phase is suggested to be the main factor affecting the conformational state and functional activity of membrane-bound proteins during aging.     

Fluorescence resonance energy transfer between donor-acceptor pair on two oligonucleotides hybridized adjacently to DNA template

We use fluorescein as the energy donor and rhodamine as the acceptor to measure the efficiency of fluorescence resonance energy transfer (FRET) in a set of hybridized DNA constructs. The two fluorophores are covalently attached via linkers to two separate oligonucleotides with fluorescein at the 3' end of one oligonucleotide and rhodamine at the 5' end or in the middle of another nucleotide. For the FRET analysis both fluorophore-labeled oligonucleotides are hybridized to adjacent sections of the same DNA template to form a three-component duplex with a one base gap between the two labeled oligonucleotides. A similar configuration is implemented for a quantitative real-time polymerase chain reaction (PCR) with LightCycler technology, where a 1-5 base separation between donor and acceptor is recommended to optimize energy transfer efficiencies. Our constructs cover donor-acceptor separations from 2 to 17 base pairs (approximately 10-70 A). The results show that, when the two fluorophores are located at close distances (less than 8 base separation), FRET efficiencies are above 80%, although there may be ground-state interactions between fluorophores when the separation is under about 6 bases. Modeling calculations are used to predict the structure of these three-component constructs. The duplex mostly retains a normal double helical structure, although slight bending may occur near the unpaired base in the DNA template. Stable and reproducible energy transfer is also observed over the distance range investigated here in real-time thermal cycling. The study identifies important parameters that determine FRET response in applications such as real-time PCR.     

Excitonic heterodimer formation in an HIV-1 oligonucleotide labeled with a donor-acceptor pair used for fluorescence resonance energy transfer

In this study, we investigated the absorbance and fluorescence properties of cTAR, the complementary DNA sequence of the transactivation response element of the HIV-1 genome, doubly end-labeled by different dyes, 5(and 6)-carboxyfluorescein (Fl) and 5(and 6)-carboxytetramethylrhodamine (TMR), frequently used in fluorescence resonance energy transfer (FRET) studies. This oligonucleotide forms a stable stem-loop structure. The absorption spectrum of this species clearly differed from that of a doubly labeled cTAR derivative in which the terminal part of the stem is melted and from an equimolecular mixture of singly labeled species. Moreover, no significant TMR fluorescence change accompanies the dramatic Fl intensity increase when the doubly labeled native cTAR was melted by temperature or annealed with its complementary sequence. Both elements suggest the formation of an H-type ground-state heterodimer between Fl and TMR that may be described by the molecular exciton model. Moreover, time-resolved fluorescence further suggests that the nonfluorescent heterodimer is in equilibrium with a small population of partially melted species showing FRET. Based on the spectral shifts associated with heterodimer formation, an interchromophore distance of 7.7 A was calculated. Both the excitonic signal and the Fl fluorescence were used as sensitive tools to monitor the temperature-mediated and HIV nucleocapsid protein-mediated annealing of cTAR with its complementary sequence.     

An improved solid-phase synthesis of resonance energy transfer fluorescent peptides and depsipeptides employing the EDANS/DABCYL donor-acceptor pair

Summary Fluorescent peptide substrates of the resonance energy transfer type, employing the donor-acceptor pair 5-[(2′-aminoethyl)amino]naphthalene sulphonic acid (EDANS) and 4-[[4′-(dimethylamino)phenyl]azo]benzoic acid (DABCYL), are widely used for continuous monitoring of the activity of various proteases. We describe here a flexible synthetic scheme which allows the synthesis of peptides having EDANS and DABCYL in any position of the sequence; the synthesis is entirely performed on solid phase with standard methods and makes use only of EDANS, DABCYL and commercially available amino acid derivatives. Moreover, we show that our scheme can be equally applied to the synthesis of EDANS/DABCYL-derivatised depsipeptides.     

An improved solid-phase synthesis of resonance energy transfer fluorescent peptides and depsipeptides employing the EDANS/DABCYL donor-acceptor pair

Fluorescent peptide substrates of the resonance energy transfer type, employing thedonor–acceptor pair 5-[(2-aminoethyl)amino]naphthalene sulphonic acid(EDANS) and 4-[[4-(dimethylamino)phenyl]azo]benzoic acid (DABCYL), are widelyused for continuous monitoring of the activity of various proteases. We describe here aflexible synthetic scheme which allows the synthesis of peptides having EDANS andDABCYL in any position of the sequence; the synthesis is entirely performed on solid phasewith standard methods and makes use only of EDANS, DABCYL and commercially availableamino acid derivatives. Moreover, we show that our scheme can be equally applied to thesynthesis of EDANS/DABCYL-derivatised depsipeptides.     

Detection of frequency resonance energy transfer pair on double-labeled microsphere and Bacillus anthracis spores by flow cytometry

Development of an ultrasensitive biosensor for biological hazards in the environment is a major need for pollutant control and for the detection of biological warfare. Fluorescence methods combined with immunodiagnostic methods are the most common. To minimize background noise, arising from the unspecific adsorption effect, we have adapted the FRET (frequency resonance energy transfer) effect to the immunofluorescence method. FRET will increase the selectivity of the diagnosis process by introducing a requirement for two different reporter molecules that have to label the antigen surface at a distance that will enable FRET. Utilizing the multiparameter capability of flow cytometry analysis to analyze the double-labeling/FRET immunostaining will lead to a highly selective and sensitive diagnostic method. This work examined the FRET interaction of fluorescence-labeled avidin molecules on biotin-coated microspheres as a model system. As target system, we have used labeled polyclonal antibodies on Bacillus anthracis spores. The antibodies used were purified immunoglobulin G (IgG) molecules raised in rabbits against B. anthracis exosoporium components. The antibodies were fluorescence labeled by a donor-acceptor chromophore pair, alexa488 as a donor and alexa594 as an acceptor. On labeling the spores with alexa488-IgG as a donor and alexa594-IgG as an acceptor, excitation at 488 nm results in quenching of the alexa-488 fluorescence (E(q) = 35%) and appearance of the alexa594 fluorescence (E(s) = 22%), as detected by flow cytometry analysis. The FRET effect leads to a further isolated gate (FL1/FL3) for the target spores compared to competitive spores such as B. thuringiensis subsp. israelensis and B. subtilis. This new approach, combining FRET labeling and flow cytometry analysis, improved the selectivity of the B. anthracis spores by a factor of 10 with respect to B. thuringiensis subsp. israelensis and a factor of 100 with respect to B. subtilis as control spores.     

Cross-linking between translationally equivalent sites on the two heads of myosin. Relationship to energy transfer results between the same pair of sites

Mercenaria myosin and scallop pure hybrid myosin possessing Mercenaria regulatory light chains were reacted with various concentrations of 4-4'-dimaleimidylstilbene-2-2'-disulfonic acid (DMSDS). Regulatory light chain homodimers are formed with great efficiency (20-50%). Dimers incorporating essential light chains were not formed upon reaction of DMSDS with Mercenaria myosin but some (less than 5%) essential light chain homodimers were obtained in the case of scallop hybrid myosin, probably occurring through relatively specific intermolecular associations within small myosin aggregates. Results were invariant, irrespective of the presence or absence of calcium and/or ATP. No radioactivity is incorporated into regulatory light chain homodimers upon post-labeling DMSDS-reacted myosin with 14C-labeled N-ethylmaleimide, irrespective of the original labeling ratio of DMSDS to myosin heads. This indicates the absence of free sulfhydryl groups in the regulatory light chain homodimer and suggests, therefore, that DMSDS links the two light chains together between translationally equivalent residues (Cys-50 of the Mercenaria regulatory light chain). These results imply that translationally equivalent sites on the two heads of myosin can come within 18 A of each other, the span of reacted DMSDS. Because energy transfer results between identical pairs of translationally equivalent sites on hybrid myosins indicated a low efficiency of energy transfer between these sites (Chantler, P.D., and Tao, T. (1986) J. Mol. Biol. 192, 87-99), it would appear that even though the two cysteines can come within 18 A of each other, their mean separation is much greater than this distance (greater than 50 A), a result consistent with a considerable flexibility of the two myosin heads with respect to each other.     

New energy transfer dyes for DNA sequencing.

We have synthesized a set of four energy transfer dyes and demonstrated their use in automated DNA sequencing. The donor dyes are the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein and the acceptor dyes are a novel set of four 4,7-dichloro-substituted rhodamine dyes which have narrower emission spectra than the standard, unsubstituted rhodamines. A rigid amino acid linker, 4-aminomethylbenzoic acid, was used to separate the dyes. The brightness of each dye in an automated sequencing instrument equipped with a dual line argon ion laser (488 and 514 nm excitation) was 2-2.5 times greater than the standard dye-primers with a 2 times reduction in multicomponent noise. The overall improvement in signal-to-noise was 4- to 5-fold. The utility of the new dye set was demonstrated by sequencing of a BAC DNA with an 80 kb insert. Measurement of the extinction coefficients and the relative quantum yields of the dichlororhodamine components of the energy transfer dyes showed their values were reduced by 20-25% compared with the dichlororhodamine dyes alone.     

Other developments regarding the concept of energy in biological systems

In order to recognize the realizability of inputs with different physical natures through a component, Yoneda's Lemma is applied. The major utility of this Lemma is when the components produce only energy. From this, it is assumed that a new material input must exist which was not recognized in the original developments in biological systems representation. Moreover, simple transfers of energy, between objects, components, and among both objects and components are developed under the generic name; energetical evolution. Thus, energetical evolution appears as anew element in the abstract representation of biological systems. These new concepts are incorporated into a new abstract diagram and a newM _β category. This paper was made possible by a Fellowship from the Consejo Nacional de Investigaciones Científicas y Técnicas of the República Argentina.     

Resonance energy transfer for assessing the molecular integrity of proteins for local delivery

It remains unclear whether the limitations to the therapeutic potential of angiogenic growth factors stem from pharmacokinetic concerns related to inadequate delivery or from a reduced sensitivity of target tissues. Here, we report a novel method using resonance energy transfer to assess the molecular integrity of proteins after local delivery. As an example, we labeled fibroblast growth factor-2 with a fluorescent donor and nonfluorescent acceptor pair, tetramethylrhodamine and QSY-7, and demonstrate in an ex vivo bovine carotid artery model that this growth factor is not limited by proteolytic constraints imposed by the tissue. Our data indicate that FGF-2 is unlikely to be degraded within the arterial wall and suggest that pharmacokinetic limitations alone cannot fully explain the muted response seen thus far in therapeutic angiogenesis. In general, resonance energy transfer may serve as a novel approach to assess the molecular integrity of protein-based therapies in local delivery.     

Using an amino acid fluorescence resonance energy transfer pair to probe protein unfolding: application to the villin headpiece subdomain and the LysM domain

Previously, we have shown that p-cyanophenylalanine (Phe CN) and tryptophan (Trp) constitute an efficient fluorescence resonance energy transfer (FRET) pair that has several advantages over commonly used dye pairs. Here, we aim to examine the general applicability of this FRET pair in protein folding-unfolding studies by applying it to the urea-induced unfolding transitions of two small proteins, the villin headpiece subdomain (HP35) and the lysin motif (LysM) domain. Depending on whether Phe CN is exposed to solvent, we are able to extract either qualitative information about the folding pathway, as demonstrated by HP35, which has been suggested to unfold in a stepwise manner, or quantitative thermodynamic and structural information, as demonstrated by LysM, which has been shown to be an ideal two-state folder. Our results show that the unfolding transition of HP35 reported by FRET occurs at a denaturant concentration lower than that measured by circular dichroism (CD) and that the loop linking helix 2 and helix 3 remains compact in the denatured state, which are consistent with the notion that HP35 unfolds in discrete steps and that its unfolded state contains residual structures. On the other hand, our FRET results on the LysM domain allow us to develop a model for extracting structural and thermodynamic parameters about its unfolding, and we find that our results are in agreement with those obtained by other methods. Given the fact that Phe CN is a non-natural amino acid and, thus, amenable to incorporation into peptides and proteins via existing peptide synthesis and protein expression methods, we believe that the FRET method demonstrated here is widely applicable to protein conformational studies, especially to the study of relatively small proteins.     

Nudged elastic band calculation of minimal energy paths for the conformational change of a GG non-canonical pair

The nudged elastic band (NEB) technique has been implemented in AMBER to calculate low-energy paths for conformational changes. A novel simulated annealing protocol that does not require an initial hypothesis for the path is used to sample low-energy paths. This was used to study the conformational change of an RNA cis Watson-Crick/Hoogsteen GG non-canonical pair, with one G syn around the glycosidic bond and the other anti. A previous solution structure, determined by NMR-constrained modeling, demonstrated that the GG pairs change from (syn)G-(anti)G to (anti)G-(syn)G in the context of duplex r(GCAGGCGUGC) on the millisecond timescale. The set of low-energy paths found by NEB show that each G flips independently around the glycosidic bond, with the anti G flipping to syn first. Guanine bases flip without opening adjacent base-pairs by protruding into the major groove, accommodated by a transient change by the ribose to C2'-exo sugar pucker. Hydrogen bonds between bases and the backbone, which lower the energetic barrier to flipping, are observed along the path. The results show the plasticity of RNA base-pairs in helices, which is important for biological processes, including mismatch repair, protein recognition, and translation. The modeling of the GG conformational change also demonstrates that NEB can be used to discover non-trivial paths for macromolecules and therefore NEB can be used as an exploratory method for predicting putative conformational change paths.     

Nanoparticle energy transfer on the cell surface

Membrane topology of receptors plays an important role in shaping transmembrane signalling of cells. Among the methods used for characterizing receptor clusters, fluorescence resonance energy transfer between a donor and acceptor fluorophore plays a unique role based on its capability of detecting molecular level (2-10 nm) proximities of receptors in physiological conditions. Recent development of biotechnology has made possible the usage of colloidal gold particles in a large size range for specific labelling of cells for the purposes of electron microscopy. However, by combining metal and fluorophore labelling of cells, the versatility of metal-fluorophore interactions opens the way for new applications by detecting the presence of the metal particles by the methods of fluorescence spectroscopy. An outstanding feature of the metal nanoparticle-fluorophore interaction is that the metal particle can enhance spontaneous emission of the fluorophore in a distance-dependent fashion, in an interaction range essentially determined by the size of the nanoparticle. In our work enhanced fluorescence of rhodamine and cyanine dyes was observed in the vicinity of immunogold nanoparticles on the surface of JY cells in a flow cytometer. The dyes and the immunogold were targetted to the cell surface receptors MHCI, MHCII, transferrin receptor and CD45 by monoclonal antibodies. The fluorescence enhancement was sensitive to the wavelength of the exciting light, the size and amount of surface bound gold beads, as well as the fluorophore-nanoparticle distance. The intensity of 90 degrees scattering of the incident light beam was enhanced by the immunogold in a concentration and size-dependent fashion. The 90 degrees light scattering varied with the wavelength of the incident light in a manner characteristic to gold nanoparticles of the applied sizes. A reduction in photobleaching time constant of the cyanine dye was observed in the vicinity of gold particles in a digital imaging microscope. Modulations of 90 degrees light scattering intensity and photobleaching time constant indicate the role of the local field in the fluorescence enhancement. A mathematical simulation based on the electrodynamic theory of fluorescence enhancement showed a consistency between the measured enhancement values, the inter-epitope distances and the quantum yields. The feasibility of realizing proximity sensors operating at distance ranges larger than that of the conventional Forster transfer is demonstrated on the surface of living cells.     

Excitation energy transfer in the light-harvesting chlorophyll

The “light-harvesting chlorophyll a/b · protein” described by Thornber has been prepared electrophoretically from spinach chloroplasts. The optical properties relevant to energy transfer have been measured in the red region (i.e. 600–700 nm). Measurements of the absorption spectrum, fluorescence excitation spectrum and excitation dependence of the fluorescence emission spectrum of this protein confirm that energy transfer from chlorophyll b to chlorophyll a is highly efficient, as is the case in concentrated chlorophyll solutions and in vivo. The excitation dependence of the fluorescence polarization shows a minimum polarization of 1.9 % at 650 nm which is the absorption maximum of chlorophyll b in the protein and rises steadily to a maximum value of 13.8 % at 695 nm, the red edge of the chlorophyll a absorption band. Analysis of these measurements shows that at least two unresolved components must be responsible for the chlorophyll a absorption maximum. Comparison of polarization measurements with those observed in vivo shows that most of the depolarization observed in vivo can take place within a single protein. Circular dichroism measurements show a doublet structure in the chlorophyll b absorption band which suggests an exciton splitting not resolved in absorption. Analysis of these data yields information about the relative orientation of the S₀→S₁ transition moments of the chlorophyll molecules within the protein.     

The medium reorganization energy for the charge transfer reactions in proteins

A low static dielectric permittivity of proteins causes the low reorganization energies for the charge transfer reactions inside them. This reorganization energy does not depend on the pre-existing intraprotein electric field. The charge transferred inside the protein interacts with its aqueous surroundings; for many globular proteins, the effect of this surroundings on the reorganization energy is comparable with the effect of reorganization of the protein itself while for the charge transfer in the middle of membrane the aqueous phase plays a minor role. Reorganization energy depends strongly on the system considered, and hence there is no sense to speak on the "protein reorganization energy" as some permanent characteristic parameter. We employed a simple algorithm for calculation of the medium reorganization energy using the numerical solution of the Poisson-Boltzmann equation. Namely, the reaction field energy was computed in two versions - all media having optical dielectric permittivity, and all the media with the static one; the difference of these two quantities gives the reorganization energy. We have calculated reorganization energies for electron transfer in cytochrome c, various ammine-ruthenated cytochromes c, azurin, ferredoxin, cytochrome c oxidase, complex of methylamine dehydrogenase with amicyanin, and for proton transfer in α-chymotrypsin. It is shown that calculation of the medium reorganization energy can be a useful tool in analysis of the mechanisms of the charge transfer reactions in proteins.     

Photolysis of the ion pair triphenylsulfonium thiophenolate induced by outersphere charge transfer excitation

The ion pair Ph₃S⁺PhS⁻ can be viewed as a sulfur-based mixed-valence system which is characterized by a PhS⁻ to Ph₃S⁺ outersphere charge transfer (OSCT) absorption at λ_max=390 nm (in CHCl₃). OSCT excitation leads to the radical pair Ph₃S/PhS which undergoes subsequent elimination and radical coupling processes with the final formation of Ph₂S. The photolysis proceeds with a quantum yield of φ=0.23 at λ_irr=436 nm.