Limitations of electronic energy transfer in the determination of lipid nanodomain sizes

Even though superresolution microscopy indicates that size of plasma membrane rafts is <20 nm, those structures have never been observed. Förster resonance energy transfer (FRET) is therefore still the most powerful optical method for characterization of such domains. In this letter we investigate relation between nanodomain affinity of a donor-acceptor (D/A) pair and the detectable nanodomain size/area. We show that probes with high affinity to the liquid-ordered (L_o) phase are required for detecting domain sizes of a few nanometers, and/or domains that occupy a few percent of the bilayer area. A combination of donors and acceptors that prefer different phases is the more favorable approach. For instance, a D/A pair with the distribution constant of donors K_D = 5 and acceptors K_A = 0.01 can resolve a broad spectrum of nanodomain sizes. On the other hand, currently available donors and acceptors that prefer the same phase, either the liquid-disordered (L_d) or L_o phase, are not so convenient for determining domain sizes <20 nm. Here the detection limits of FRET experiments employing several commonly used D/A pairs have been investigated.     

Perfringolysin O association with ordered lipid domains: implications for transmembrane protein raft affinity

Upon interaction with cholesterol, perfringolysin O (PFO) inserts into membranes and forms a rigid transmembrane (TM) β-barrel. PFO is believed to interact with liquid ordered lipid domains (lipid rafts). Because the origin of TM protein affinity for rafts is poorly understood, we investigated PFO raft affinity in vesicles having coexisting ordered and disordered lipid domains. Fluorescence resonance energy transfer (FRET) from PFO Trp to domain-localized acceptors indicated that PFO generally has a raft affinity between that of LW peptide (low raft affinity) and cholera toxin B (high raft affinity) in vesicles containing ordered domains rich in brain sphingomyelin or distearoylphosphatidylcholine. FRET also showed that ceramide, which increases exposure of cholesterol to water and thus displaces it from rafts, does not displace PFO from ordered domains. This can be explained by shielding of PFO-bound cholesterol from water. Finally, FRET showed that PFO affinity for ordered domains was higher in its non-TM (prepore) form than in its TM form, demonstrating that the TM portion of PFO interacts unfavorably with rafts. Microscopy studies in giant unilamellar vesicles confirmed that PFO exhibits intermediate raft affinity, and showed that TM PFO (but not non-TM PFO) concentrated at the edges of liquid ordered domains. These studies suggest that a combination of binding to raft-associating molecules and having a rigid TM structure that is unable to pack well in a highly ordered lipid environment can control TM protein domain localization. To accommodate these constraints, raft-associated TM proteins in cells may tend to locate within liquid disordered shells encapsulated within ordered domains.     

FRET detection of cellular alpha4-integrin conformational activation

Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to undergo conformational changes upon activation. Here, different affinity states were generated by exposing alpha4-integrins to divalent ions or by inside-out activation using a chemokine receptor. We probed the dynamic structural transformation of the integrin on live cells using fluorescence resonance energy transfer (FRET) between a peptide donor, which specifically binds to the alpha4-integrin, and octadecyl rhodamine B acceptors incorporated into the plasma membrane. We analyzed the data using a model that describes FRET between a random distribution of donors and acceptors in an infinite plane. The distance of closest approach was found to vary with the affinity of the integrin. The change in distance of closest approach was approximately 50 A between resting and Mn2+ activated receptors and approximately 25 A after chemokine activation. We used confocal microscopy to probe the lateral organization of donors and acceptors subsequent to integrin activation. Taken together, FRET and confocal results suggest that changes in FRET efficiencies are primarily due to the vertical extension of the integrin. The coordination between the extension of alpha4-integrin and its affinity provides a mechanism for Dembo's catch-bond concept.     

Chromatin nanoscale compaction in live cells visualized by acceptor‐to‐donor ratio corrected Förster resonance energy transfer between DNA dyes

@Chromatin nanoscale architecture in live cells can be studied by Förster resonance energy transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system, the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor‐to‐donor ratio rather than distance. Here, to get a FRET level independent of the acceptor‐to‐donor ratio, we combine fluorescence lifetime imaging detection of FRET with a normalization of the FRET level to a pixel‐wise estimation of the acceptor‐to‐donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that this acceptor‐to‐donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser‐induced microirradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.      

Anomalous Surplus Energy Transfer Observed with Multiple FRET Acceptors

Background

Förster resonance energy transfer (FRET) is a mechanism where energy is transferred from an excited donor fluorophore to adjacent chromophores via non-radiative dipole-dipole interactions. FRET theory primarily considers the interactions of a single donor-acceptor pair. Unfortunately, it is rarely known if only a single acceptor is present in a molecular complex. Thus, the use of FRET as a tool for measuring protein-protein interactions inside living cells requires an understanding of how FRET changes with multiple acceptors. When multiple FRET acceptors are present it is assumed that a quantum of energy is either released from the donor, or transferred in toto to only one of the acceptors present. The rate of energy transfer between the donor and a specific acceptor (k_D→A) can be measured in the absence of other acceptors, and these individual FRET transfer rates can be used to predict the ensemble FRET efficiency using a simple kinetic model where the sum of all FRET transfer rates is divided by the sum of all radiative and non-radiative transfer rates.

Methodology/Principal Findings

The generality of this approach was tested by measuring the ensemble FRET efficiency in two constructs, each containing a single fluorescent-protein donor (Cerulean) and either two or three FRET acceptors (Venus). FRET transfer rates between individual donor-acceptor pairs within these constructs were calculated from FRET efficiencies measured after systematically introducing point mutations to eliminate all other acceptors. We find that the amount of energy transfer observed in constructs having multiple acceptors is significantly greater than the FRET efficiency predicted from the sum of the individual donor to acceptor transfer rates.

Conclusions/Significance

We conclude that either an additional energy transfer pathway exists when multiple acceptors are present, or that a theoretical assumption on which the kinetic model prediction is based is incorrect.     

The Impact of Heterogeneity and Dark Acceptor States on FRET: Implications for Using Fluorescent Protein Donors and Acceptors

Förster resonance energy transfer (FRET) microscopy is widely used to study protein interactions in living cells. Typically, spectral variants of the Green Fluorescent Protein (FPs) are incorporated into proteins expressed in cells, and FRET between donor and acceptor FPs is assayed. As appreciable FRET occurs only when donors and acceptors are within 10 nm of each other, the presence of FRET can be indicative of aggregation that may denote association of interacting species. By monitoring the excited-state (fluorescence) decay of the donor in the presence and absence of acceptors, dual-component decay analysis has been used to reveal the fraction of donors that are FRET positive (i.e., in aggregates)._However, control experiments using constructs containing both a donor and an acceptor FP on the same protein repeatedly indicate that a large fraction of these donors are FRET negative, thus rendering the interpretation of dual-component analysis for aggregates between separately donor-containing and acceptor-containing proteins problematic. Using Monte-Carlo simulations and analytical expressions, two possible sources for such anomalous behavior are explored: 1) conformational heterogeneity of the proteins, such that variations in the distance separating donor and acceptor FPs and/or their relative orientations persist on time-scales long in comparison with the excited-state lifetime, and 2) FP dark states.     

Global hairpin folding of tau in solution

The microtubule-associated protein tau stabilizes microtubules in its physiological role, whereas it forms insoluble aggregates (paired helical filaments) in Alzheimer's disease. Soluble tau is considered a natively unfolded protein whose residual folding and intramolecular interactions are largely undetermined. In this study, we have applied fluorescence resonance energy transfer (FRET) and electron paramagnetic resonance (EPR) to examine the proximity and flexibility of tau domains and the global folding. FRET pairs spanning the tau molecule were created by inserting tryptophans (donor) and cysteines (labeled with IAEDANS as an acceptor) by site-directed mutagenesis. The observed FRET distances were significantly different from those expected for a random coil. Notably, the C-terminal end of tau folds over into the vicinity of the microtubule-binding repeat domain, the N-terminus remains outside the FRET distance of the repeat domain, yet both ends of the molecule approach one another. The interactions between the domains were obliterated by denaturation in GdnHCl. Paramagnetic spin-labels attached in various domains of tau were analyzed by EPR and exhibited a high mobility throughout. The data indicate that tau retains some global folding even in its "natively unfolded" state, combined with the high flexibility of the chain.     

Effect of membrane microheterogeneity and domain size on fluorescence resonance energy transfer

Studies of multicomponent membranes suggest lateral inhomogeneity in the form of membrane domains, but the size of small (nanoscale) domains in situ cannot be determined with current techniques. In this article, we present a model that enables extraction of membrane domain size from time-resolved fluorescence resonance energy transfer (FRET) data. We expand upon a classic approach to the infinite phase separation limit and formulate a model that accounts for the presence of disklike domains of finite dimensions within a two-dimensional infinite planar bilayer. The model was tested against off-lattice Monte Carlo calculations of a model membrane in the liquid-disordered (l(d)) and liquid-ordered (l(o)) coexistence regime. Simulated domain size was varied from 5 to 50 nm, and two fluorophores, preferentially partitioning into opposite phases, were randomly mixed to obtain the simulated time-resolved FRET data. The Monte Carlo data show clear differences in the efficiency of energy transfer as a function of domain size. The model fit of the data yielded good agreement for the domain size, especially in cases where the domain diameter is <20 nm. Thus, data analysis using the proposed model enables measurement of nanoscale membrane domains using time-resolved FRET.     

Covalent labeling of cell-surface proteins for in-vivo FRET studies

Fluorescence resonance energy transfer (FRET) is a powerful technique to reveal interactions between membrane proteins in live cells. Fluorescence labeling for FRET is typically performed by fusion with fluorescent proteins (FP) with the drawbacks of a limited choice of fluorophores, an arduous control of donor-acceptor ratio and high background fluorescence arising from intracellular FPs. Here we show that these shortcomings can be overcome by using the acyl carrier protein labeling technique. FRET revealed interactions between cell-surface neurokinin-1 receptors simultaneously labeled with a controlled ratio of donors and acceptors. Moreover, using FRET the specific binding of fluorescent agonists could be monitored.     

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.     

Cerulean, Venus, and VenusY67C FRET reference standards

F?rster's resonance energy transfer (FRET) can be used to study protein-protein interactions in living cells. Numerous methods to measure FRET have been devised and implemented; however, the accuracy of these methods is unknown, which makes interpretation of FRET efficiency values difficult if not impossible. This problem exists due to the lack of standards with known FRET efficiencies that can be used to validate FRET measurements. The advent of spectral variants of green fluorescent protein and easy access to cell transfection technology suggests a simple solution to this problem: the development of genetic constructs with known FRET efficiencies that can be replicated with high fidelity and freely distributed. In this study, fluorescent protein constructs with progressively larger separation distances between donors and acceptors were generated and FRET efficiencies were measured using fluorescence lifetime spectroscopy, sensitized acceptor emission, and spectral imaging. Since the results from each method were in good agreement, the FRET efficiency value of each construct could be determined with high accuracy and precision, thereby justifying their use as standards.     

Multiplexed FRET to image multiple signaling events in live cells

We report what to our knowledge is a novel approach for simultaneous imaging of two different Förster resonance energy transfer (FRET) sensors in the same cell with minimal spectral cross talk. Previous methods based on spectral ratiometric imaging of the two FRET sensors have been limited by the availability of suitably bright acceptors for the second FRET pair and the spectral cross talk incurred when measuring in four spectral windows. In contrast to spectral ratiometric imaging, fluorescence lifetime imaging (FLIM) requires measurement of the donor fluorescence only and is independent of emission from the acceptor. By combining FLIM-FRET of the novel red-shifted TagRFP/mPlum FRET pair with spectral ratiometric imaging of an ECFP/Venus pair we were thus able to maximize the spectral separation between our chosen fluorophores while at the same time overcoming the low quantum yield of the far red acceptor mPlum. Using this technique, we could read out a TagRFP/mPlum intermolecular FRET sensor for reporting on small Ras GTP-ase activation in live cells after epidermal growth factor stimulation and an ECFP/Venus Cameleon FRET sensor for monitoring calcium transients within the same cells. The combination of spectral ratiometric imaging of ECFP/Venus and high-speed FLIM-FRET of TagRFP/mPlum can thus increase the spectral bandwidth available and provide robust imaging of multiple FRET sensors within the same cell. Furthermore, since FLIM does not require equal stoichiometries of donor and acceptor, this approach can be used to report on both unimolecular FRET biosensors and protein-protein interactions with the same cell.     

The inhibition of fluorescence resonance energy transfer between quantum dots for glucose assay

Fluorescence resonance energy transfer (FRET) between two quantum dots of different sizes causes fluorescence quenching. Hereby a binding site pre-blocking approach is proposed to avoid this effect. Pre-binding of glucose on the donor occupies the binding sites and thus blocks resonance energy transfer between the two quantum dots, protecting the fluorescence from being quenched. A glucose assay is developed based on this approach. The glucose content is correlated with the fluorescence difference in the absence and in the presence of glucose. In practice, Green QDs-Con A conjugates are used as donors and Red QDs-NH(2)-glu conjugates as acceptors to form FRET system. The inhibition of fluorescence quenching is then measured in the presence of glucose. A linear calibration graph is achieved within 0.1-2.0 mmolL(-1), along with a detection limit of 0.03 mmolL(-1) and a RSD of 2.1% (1.0 mmolL(-1)). 91-105% of glucose in serum and urine samples is recovered. It is worth mentioning that the present glucose assay approach also generates a fluorescence chromatic difference imaging, and the color display clearly identifies the glucose contents by visual detection with a distinguishing ability of ca. 0.5 mmolL(-1). The present approach can potentially be used for the clinical determination of glucose in biological samples which can be further developed into a glucose sensor.     

Proximity of the protein moiety of a GPI-anchored protein to the membrane surface: a FRET study

GPI-anchored proteins are ubiquitous on the eukaryotic cell surface, where they are involved in a variety of functions ranging from adhesion to enzymatic catalysis. Indirect evidence suggests that the GPI anchor may hold the protein close to the plasma membrane; however, there is a lack of direct information on the proximity of the protein portion of GPI-anchored proteins to the bilayer surface. The present study uses fluorescence resonance energy transfer (FRET) to address this important problem. The GPI-anchored ectoenzyme placental alkaline phosphatase (PLAP) was purified from a plasma membrane extract of human placental microsomes without the use of butanol. The protein was fluorescently labeled at the N-terminus with 7-(dimethylamino)coumarin-4-acetic acid succinimidyl ester (DMACA-SE) or Oregon Green 488 succinimidyl ester (OG488-SE), and each was reconstituted by detergent dilution into defined lipid bilayer vesicles containing an increasing mole fraction of a fluorescent lipid probe. The fluorescence of the labeled PLAP donors was quenched in a concentration-dependent manner by the lipid acceptors. The energy transfer data were analyzed using an approach that describes FRET between a uniform distribution of donors and acceptors in an infinite plane. The distance of closest approach between the protein moiety of PLAP and the lipid-water interfacial region of the bilayer was estimated to be smaller than 10-14 A. This indicates that the protein portion of PLAP is located very close to the lipid bilayer, possibly resting on the surface. This contact may allow transmission of structural changes from the membrane surface to the protein, which could influence the behavior and catalytic properties of GPI-anchored proteins.     

Coclustering of ErbB1 and ErbB2 Revealed by FRET-Sensitized Acceptor Bleaching

Classical theory states that ligand binding induces the dimerization of ErbB proteins, leading to their activation. Although we and other investigators have shown the existence of preformed homoclusters of ErbB receptors and analyzed their composition, the stoichiometry of their heteroclusters has not been quantitatively described. Here, we report the development of the fluorescence resonance energy transfer (FRET)-sensitized acceptor bleaching (FSAB) technique to quantitate the ratio of ErbB1 and ErbB2 in their heteroclusters. In FSAB, photolabile acceptors within FRET distance from photostable donors are excited and photobleached by FRET, and the fraction of acceptors that are participating in FRET is determined. In quiescent SKBR-3 breast cancer cells, ∼35% of ErbB1 and ∼10% of ErbB2 have been found in heteroclusters. Epidermal growth factor (ligand of ErbB1) increased the fraction of ErbB2 heteroclustering with ErbB1, whereas the ratio of heteroclustered ErbB1 did not change significantly. The fractions of heteroclustered ErbB1 and ErbB2 were independent of their expression levels, indicating that the formation of these clusters is not driven by the law of mass action. In contrast, the FRET efficiency depended on the donor/acceptor ratio as expected. We present a model in which preformed receptor clusters are rearranged upon ligand stimulation, and report that the composition of these clusters can be quantitatively described by the FSAB technique.     

Imaging FRET between spectrally similar GFP molecules in single cells

Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca(2+) ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.     

A rigorous experimental framework for detecting protein oligomerization using bioluminescence resonance energy transfer

Bioluminescence resonance energy transfer (BRET), which relies on nonradiative energy transfer between luciferase-coupled donors and GFP-coupled acceptors, is emerging as a useful tool for analyzing the quaternary structures of cell-surface molecules. Conventional BRET analyses are generally done at maximal expression levels and single acceptor/donor ratios. We show that under these conditions substantial energy transfer arises from random interactions within the membrane. The dependence of BRET efficiency on acceptor/donor ratio at fixed surface density, or expression level at a defined acceptor/donor ratio, can nevertheless be used to correctly distinguish between well-characterized monomeric and oligomeric proteins, including a very weak dimer. The pitfalls associated with the nonrigorous treatment of BRET data are illustrated for the case of G protein-coupled receptors (GPCRs) proposed to form homophilic and/or mixed oligomers on the basis of previous, conventional BRET experiments.     

dSPRINT: predicting DNA,RNA, ion,peptide and small molecule interaction sites within protein domains

Domains are instrumental in facilitating protein interactions with DNA, RNA, small molecules, ions and peptides. Identifying ligand-binding domains within sequences is a critical step in protein function annotation, and the ligand-binding properties of proteins are frequently analyzed based upon whether they contain one of these domains. To date, however, knowledge of whether and how protein domains interact with ligands has been limited to domains that have been observed in co-crystal structures; this leaves approximately two-thirds of human protein domain families uncharacterized with respect to whether and how they bind DNA, RNA, small molecules, ions and peptides. To fill this gap, we introduce dSPRINT, a novel ensemble machine learning method for predicting whether a domain binds DNA, RNA, small molecules, ions or peptides, along with the positions within it that participate in these types of interactions. In stringent cross-validation testing, we demonstrate that dSPRINT has an excellent performance in uncovering ligand-binding positions and domains. We also apply dSPRINT to newly characterize the molecular functions of domains of unknown function. dSPRINT’s predictions can be transferred from domains to sequences, enabling predictions about the ligand-binding properties of 95% of human genes. The dSPRINT framework and its predictions for 6503 human protein domains are freely available at http://protdomain.princeton.edu/dsprint.     

High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes

"Lipid rafts" enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.     

Structure of a novel Bence-Jones protein (Rhe) fragment at 1.6 A resolution

The crystal structure of Rhe, a lambda-type Bence-Jones protein fragment, has been solved and refined to a resolution of 1.6 A. A model fragment consisting of the complete variable domain and the first three residues of the constant domain yields a crystallographic residual RF value of 0.149. The protein exists as a dimer both in solution and in the crystals. Although the "immunoglobulin fold" is generally preserved in the structure, there are significant differences in both the monomer conformation and in the mode of association of monomers into dimers, when compared to other known Bence-Jones proteins or Fab fragments. The variations in conformation within monomers are particularly significant as they involve non-hypervariable residues, which previously were believed to be part of a "structurally invariant" framework common to all immunoglobulin variable domains. The novel mode of dimerization is equally important, as it can result in combining site shapes and sizes unobtainable with the conventional mode of dimerization. A comparison of the structure with other variable domain dimers reveals further that the variations within monomers and between domains in the dimer are coupled. Some possible functional implications revealed by this coupling are greater variability, induced fitting of the combining site to better accommodate antigenic determinants, and a mechanism for relaying binding information from one end of the variable domain dimer to the other. In addition to providing the most accurate atomic parameters for an immunoglobulin domain yet obtained, the high resolution and extensive refinement resulted in identification of several tightly bound water molecules in key structural positions. These water molecules may be regarded as integral components of the protein. Other water molecules appear to be required to stabilize the novel conformation.