Forster Resonance Energy Transfer Experiments Research Articles

Quantitative Interpretation of FRET Experiments via Molecular Simulation: Force Field and Validation

Molecular simulation is a valuable and complementary tool that may assist with the interpretation of single-molecule Förster resonance energy transfer (FRET) experiments, if the energy function is of sufficiently high quality. Here we present force-field parameters for one of the most common pairs of chromophores used in experiments, AlexaFluor 488 and 594. From microsecond molecular-dynamics simulations, we are able to recover both experimentally determined equilibrium constants and association/dissociation rates of the chromophores with free tryptophan, as well as the decay of fluorescence anisotropy of a labeled protein. We find that it is particularly important to obtain a correct balance of solute-water interactions in the simulations in order to faithfully capture the experimental anisotropy decays, which provide a sensitive benchmark for fluorophore mobility. Lastly, by a combination of experiment and simulation, we address a potential complication in the interpretation of experiments on polyproline, used as a molecular ruler for FRET experiments, namely the potential association of one of the chromophores with the polyproline helix. Under conditions where simulations accurately capture the fluorescence anisotropy decay, we find at most a modest, transient population of conformations in which the chromophores associate with the polyproline. Explicit calculation of FRET transfer efficiencies for short polyprolines yields results in good agreement with experiment. These results illustrate the potential power of a combination of molecular simulation and experiment in quantifying biomolecular dynamics.

Transition Metal Ion FRET Measurements in Plasma Membrane Sheets Prepared by Deroofing Cultured Mammalian Cells

Since the discovery of Forster resonance energy transfer (FRET) in the 1940s, its implementation to measure inter- and intramolecular distances has reached wide-spread use in the biological sciences. Because the technique requires labeling of a donor and acceptor site with two different spectroscopically active molecules, a prominent difficulty with biological samples has been how to label with specificity and, therefore, unambigouisly detect and assign the signal contribution from each site. Here we show that transition metal ion FRET (tmFRET) experiments using plasma membrane sheets prepared from deroofed cells can be used as an orthogonal labeling scheme with an unambiguous assignment of the FRET pair signal. As a FRET donor, we used octadecyl rhodamine B (R18), which incorporated into the plasma membrane of deroofed cultured cells. To measure distances on the length scale relevant to membrane proteins, we used the transition metal ion Co2+ as an acceptor, which, together with R18, gives a R0 value of 14.9 A. Although the quenching by Co2+ of rhodamine B in solution is of low affinity (>10 mM), 100 μM Co2+ caused appreciable quenching of R18 incorporated into plasma membrane sheets. By first introducing a synthetic fatty acid with a metal-chelating head group, appreciable quenching was observed with less than 1 μM Co2+. Comparison of tmFRET experimental measurements to expected theoretical values convincingly demonstrate that these experiments provide information about the distance of closest approach between donor and acceptor (Fung and Stryer, 1978). These experiments provide proof-of-priniciple for measurements of biologically relevant distances in native membranes. We anticipate that FRET experiments on plasma membrane sheets will offer a unique opportunity to explore protein-lipid interactions and discuss several possible future directions for this technique.

Construction, imaging, and analysis of FRET-based tension sensors in living cells.

Due to an increased appreciation for the importance of mechanical stimuli in many biological contexts, an interest in measuring the forces experienced by specific proteins in living cells has recently emerged. The development and use of Förster resonance energy transfer (FRET)-based molecular tension sensors has enabled these types of studies and led to important insights into the mechanisms those cells utilize to probe and respond to the mechanical nature of their surrounding environment. The process for creating and utilizing FRET-based tension sensors can be divided into three main parts: construction, imaging, and analysis. First we review several methods for the construction of genetically encoded FRET-based tension sensors, including restriction enzyme-based methods as well as the more recently developed overlap extension or Gibson Assembly protocols. Next, we discuss the intricacies associated with imaging tension sensors, including optimizing imaging parameters as well as common techniques for estimating artifacts within standard imaging systems. Then, we detail the analysis of such data and describe how to extract useful information from a FRET experiment. Finally, we provide a discussion on identifying and correcting common artifacts in the imaging of FRET-based tension sensors.

Rise-time of FRET-acceptor fluorescence tracks protein folding.

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxinʼs complex folding pathway.

Molecular dynamics simulation of configurational ensembles compatible with experimental FRET efficiency data through a restraint on instantaneous FRET efficiencies.

Förster resonance energy transfer (FRET) measurements are widely used to investigate (bio)molecular interactions or/and association. FRET efficiencies, the primary data obtained from this method, give, in combination with the common assumption of isotropic chromophore orientation, detailed insight into the lengthscale of molecular phenomena. This study illustrates the application of a FRET efficiency restraint during classical atomistic molecular dynamics simulations of a mutant mastoparan X peptide in either water or 7 M aqueous urea. The restraint forces acting on the donor and acceptor chromophores ensure that the sampled peptide configurational ensemble satisfies the experimental primary data by modifying interchromophore separation and chromophore transition dipole moment orientations. By means of a conformational cluster analysis, it is seen that indeed different configurational ensembles may be sampled without and with application of the restraint. In particular, while the FRET efficiency and interchromophore distances monitored in an unrestrained simulation may differ from the experimentally-determined values, they can be brought in agreement with experimental data through usage of the FRET efficiency restraining potential. Furthermore, the present results suggest that the assumption of isotropic chromophore orientation is not always justified. The FRET efficiency restraint allows the generation of configurational ensembles that may not be accessible with unrestrained simulations, and thereby supports a meaningful interpretation of experimental FRET results in terms of the underlying molecular degrees of freedom. Thus, it offers an additional tool to connect the realms of computer and wet-lab experimentation.

Implementation of transportation distance for analyzing FLIM and FRET experiments.

Analysis of fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) experiments in living cells is usually based on mean lifetimes computations. However, these mean lifetimes can induce misinterpretations. We propose in this work the implementation of the transportation distance for FLIM and FRET experiments in vivo. This non-fitting indicator, which is easy to compute, reflects the similarity between two distributions and can be used for pixels clustering to improve the estimation of the FRET parameters. We study the robustness and the discriminating power of this transportation distance, both theoretically and numerically. In addition, a comparison study with the largely used mean lifetime differences is performed. We finally demonstrate practically the benefits of the transportation distance over the usual mean lifetime differences for both FLIM and FRET experiments in living cells.

FRET: signals hidden within the noise.

F€ orster resonance energy transfer (FRET) is a physical phenomenon that has been of incredible utility to the scientific community, enabling the study of a wide range of nanoscale, distance-dependent measurements. FRET occurs when energy is nonradiatively transferred from an excited fluorophore (the donor fluorophore) to a second fluorophore in near proximity (the acceptor fluorophore) (1,2). Because the rate at which energy is transferred from the donor to the acceptor fluorophore depends on the intermolecular distance raised to the sixth power, FRET provides a highly sensitive measure of intermolecular spacing (2,3). The key parameter in FRET measurements is the FRET efficiency. FRET efficiency is most often described as the ratio of the number of quantized energy events (sometimes referred to as virtual photons (4)) transferred from the donor fluorophore to the acceptor fluorophore, divided by the total number of quantized energy events (photons) absorbed by the donor fluorophore (5). Using appropriate measurement and computational techniques, FRET efficiency represents a quantifiable value describing the intermolecular spacing and orientation within a sample. For fluorophores with fixed intermolecular spacing, the value of the measured FRET efficiency has been shown to be relatively constant, regardless of the measurement technique used (6). Hence, FRET reporters can be used to provide absolute quantitative data describing interand intramolecular interactions. Computing the FRET efficiency, whether in fluorescence microscopy or flow cytometry measurements, typically requires a ratiometric calculation (7). Because FRET efficiencies often range from 20 to 60% for biological reporters, the fluorescence emission from the acceptor fluorophore, when excited via energy transfer, usually provides lower signal strength than measurements of acceptor fluorophore emission resulting from direct excitation. Performing ratiometric calculations using relatively weak signals as the inputs can result in compounding error propagation (8). Thus, while FRET, as a tool, offers the ability to probe molecular distances within images or flow cytometry data, it also introduces substantial limitations in sensitivity and variance, due to low signal strength. Low signal strength may also limit spatial and temporal resolution. When performing FRET experiments, trade-offs must usually be made between signal strength (accuracy of the FRET efficiency calculation), spatial resolution, and temporal

Magnetoliposomes based on nickel/silica core/shell nanoparticles: Synthesis and characterization

In the present work, nickel magnetic nanoparticles with diameters lower than 100 nm, with and without silica shell, were synthesized by microheterogeneous templating. The magnetic properties of the nanoparticles show a typical ferromagnetic behavior with a coercive field of 80 Oe. Dry magnetoliposomes (DMLs) with diameter between 58 nm and 76 nm were obtained from the synthesis of nanoparticles in the presence of a lipid or surfactant layer, and aqueous magnetoliposomes (AMLs) were obtained by encapsulation of the nanoparticles in liposomes. FRET (Förster resonance energy transfer) experiments were performed to study the non-specific interactions between aqueous magnetoliposomes and giant unilamellar vesicles (GUVs), as models of cell membranes. It was possible to detect membrane fusion between GUVs and AMLs containing both NBD-C6-HPC (donor) and the dye Nile Red (acceptor).

Internally labeled Cy3/Cy5 DNA constructs show greatly enhanced photo-stability in single-molecule FRET experiments

DNA constructs labeled with cyanine fluorescent dyes are important substrates for single-molecule (sm) studies of the functional activity of protein–DNA complexes. We previously studied the local DNA backbone fluctuations of replication fork and primer–template DNA constructs labeled with Cy3/Cy5 donor–acceptor Förster resonance energy transfer (FRET) chromophore pairs and showed that, contrary to dyes linked ‘externally’ to the bases with flexible tethers, direct ‘internal’ (and rigid) insertion of the chromophores into the sugar-phosphate backbones resulted in DNA constructs that could be used to study intrinsic and protein-induced DNA backbone fluctuations by both smFRET and sm Fluorescent Linear Dichroism (smFLD). Here we show that these rigidly inserted Cy3/Cy5 chromophores also exhibit two additional useful properties, showing both high photo-stability and minimal effects on the local thermodynamic stability of the DNA constructs. The increased photo-stability of the internal labels significantly reduces the proportion of false positive smFRET conversion ‘background’ signals, thereby simplifying interpretations of both smFRET and smFLD experiments, while the decreased effects of the internal probes on local thermodynamic stability also make fluctuations sensed by these probes more representative of the unperturbed DNA structure. We suggest that internal probe labeling may be useful in studies of many DNA–protein interaction systems.

The Cardiac Troponin T R92L Hcm Mutation Alters Cardiac Troponin I Dynamics and PKA Phosphorylation Potential

Mutations in cardiac troponin T (cTnT) have been linked to hypertrophic cardiomyopathy (HCM). Previous experiments in our lab utilizing a novel cTnT R92L transgenic mouse model revealed an allosterically mediated decrease in the PKA phosphorylation potential of cardiac troponin I (cTnI). We hypothesized the cTnT R92L mutation causes a change in the dynamics of the N-terminal extension, hindering the availability of PKA substrates S23/S24 on cTnI. In the current study this hypothesis was tested both in silico and in vitro. First, molecular dynamics (MD) simulations using our all-atom thin filament model in the Ca2+ saturated state were performed. MD predicted an approximately 7A narrowing between the N-terminal extension of cTnI and cardiac troponin C (cTnC). To experimentally evaluate the in-silico predictions, Forster Resonance Energy Transfer (FRET) experiments were conducted on fully reconstituted thin filaments. The cTnI was labeled with the FRET donor AEDANS at three sites on the N-terminal extension of cTnI. The acceptor DDPM, was labeled on six sites on cTnC. Steady state and time resolved FRET experiments were performed in the presence and absence of the TnT R92L mutation. Both theoretical and experimental approaches revealed that in the presence of the cTnT R92L mutation the dynamics of cTnI was allosterically altered. The N-terminal extension of cardiac troponin I moved closer to the N-domain of cTnC. This anharmonic fluctuation likely hinders the availability of the PKA substrate sites and leads to the observed blunting of the beta-adrenergic response in animal models and human patients.

Commentary: Assessing FRET using spectral techniques

The manuscript “Assessing FRET using spectral techniques” by Leavesley et al. (page 898 in this issue) addresses Förster resonance energy transfer (FRET) between organic molecules due to an interaction of optical transition dipole moments. Although occurring in nature for more than a billion of years, a theoretical description of this mechanism has only been given in 1948 1. Since that time energy transfer spectroscopy has been used to probe molecular distances and mechanisms in the nanometre range 2-4, profiting from the fact that the energy transfer rate depends on the sixth power of molecular distance (kET ∼ r−6) and is, therefore, a very sensitive parameter. FRET experiments gained considerable importance, when in the 1990s green fluorescent protein [GFP, naturally occurring in the jellyfish Aequorea Victoria, 5] and its mutants could be cloned and fused with almost any protein of a cell. So, nonradiative energy transfer from cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP) or from GFP to red fluorescent protein was used to probe changes of molecular conformation, for example, upon binding of calcium 6, or interactions between adjacent molecules, for example, protein−protein interactions playing an important role in regulation of apoptosis 7 or in pathogenesis of M. Alzheimer and further neurodegenerative diseases 8, 9. Due to these experimental restrictions (and pronounced costs) of time-resolving FRET experiments, spectral techniques and algorithms upon stationary optical excitation still play an important role, as outlined in the manuscript by Leavesley et al. (page 898 in this issue). Here, the authors used various techniques and algorithms based on up to three filter sets (e.g., for measurement of FRET efficiency at variable donor concentration) as well as spectral unmixing. Using a probe, where CFP and YFP are linked by a cAMP binding protein, they measured FRET efficiency in cultivated cells as a function of cAMP concentration. Although a two-filter set approach was already able to describe FRET efficiency with rather low coefficients of variation, spectrally resolved experiments provided improved results. The authors concluded that hyperspectral confocal microscopy in combination with linear unmixing, cell segmentation, and quantitative image analysis was appropriate for FRET experiments in single cells. I would like to share this opinion, emphasizing that in an ideal case spectral and time-resolving FRET experiments might be combined. This holds in particular, if acceptor fluorescence is low 8, or if donor and acceptor fluorescence are spectrally indiscernible [Homo FRET, 11]. During the last years resolution in optical microscopy has been improved considerably. Starting from a value around 200 nm, as resulting from Abbé's diffraction theory, values of 10−30 nm were meanwhile obtained by super-localization microscopy based on single molecule detection 12, 13 or by stimulated emission depletion microscopy [STED, 14]. Resolution in FRET experiments still goes below these values and permits to measure molecular distances of 5 nm and less. In addition, high irradiance as needed for STED (and to some extent also for single molecule detection) and risking severe cell damage can be avoided in FRET experiments. Furthermore, FRET is not restricted to microscopy and not limited to adherent samples, but can be combined with any other method, for example, flow cytometry 15.

Förster resonance energy transfer studies of calmodulin produced by native protein ligation reveal inter‐domain electrostatic repulsion

This study explores the influence of long-range intra-protein electrostatic interactions on the conformation of calmodulin in solution. Ensemble Förster resonance energy transfer (FRET) is measured for calmodulin with a fluorophore pair incorporated specifically with a donor at residue 17 and an acceptor at position 117. This construct was generated by a combination of solid phase peptide synthesis, cloning, expression and native chemical ligation. This labelling method has not previously been used with calmodulin and represents a convenient method for ensuring the explicit positioning of the fluorophores. The ensemble FRET experiments reveal significant electrostatic repulsion between the globular domains in the calcium-free protein. At low salt, calmodulin has a relatively extended conformation and the distance between the domains is further increased by denaturation, by heat or by non-ionic denaturants. The repulsion between domains is screened by salt and is also diminished by calcium binding, which changes the protein net charge from -23 to -15. Compared with the calcium-free form at low salt, the FRET efficiency for the calcium-bound form has, on average, increased 10-fold. The conformation of the calcium form is insensitive to salt screening. These results imply that when the two globular domains of calmodulin interact with target, there is no significant free energy penalty due to electrostatic interactions.

Accurate Intramolecular Distances by Single Molecule Confocal Spectroscopy: A Monte Carlo Markov Chain Analysis of Fluorescence Data from Freely Diffusing Biomolecules

Forster Resonance Energy Transfer (FRET) is a powerful technique for studying the conformational dynamics of biological systems at the level of single molecules. FRET experiments on single molecules (smFRET) in solution have the potential to recover accurate intramolecular distances. However, established methods for event selection and de-noising of smFRET data, are ad hoc and cause systematic biases in the results obtained.We introduce a novel method for the analysis of smFRET data, suitable for the study of protein folding or molecular association. The technique is based on a simple stochastic model of the physical FRET process. Based on this model, we devise a Bayesian inference technique to infer directly all key features of a smFRET dataset, using Markov Chain Monte Carlo (MCMC) sampling methods. The proposed technique is computationally fast, requires only a few minutes of data collection, yields reliable confidence intervals, and requires no subjective choice of parameters for de-noising.We evaluate the technique's performance using a combination of synthetic and experimental data, demonstrating that the method:i. effectively identifies fluorescence events;ii. distinguishes noise and fluorescence contributions to fluorescence events;iii. infers simultaneously the rate parameters for the noise distribution, the FRET efficiencies, and relative sizes of one or more fluorescent populations.We validate the technique on smFRET data from mixtures of well characterised dual-labelled DNA duplexes. Our analysis method is able to infer both the mean FRET efficiencies of multiple FRET populations and the relative sizes of these populations. Results will be presented demonstrating that our Bayesian inference technique obtains accurate intramolecular distance information from smFRET experiments, whereas established techniques fail.

The Binding Parameters of Integration Host Factor to the Lambda Phage Cosi1 Site and Holliday Junction DNA

Integration Host Factor (IHF) is a heterodimeric DNA-binding protein produced by E.coli. IHF interacts with the minor groove and phosphodiester backbone of the DNA to create sharp bends in the DNA and is often referred to as an architectural protein. Although the protein binds sequence-specifically, it also recognizes DNA conformation. Prokaryotic cellular processes involving IHF include viral integration, replication and activation of gene expression. We have recently shown that IHF exhibits high binding affinity for the Holliday or four-way junction DNA (4WJ), an intermediate in homologous recombination where strands from different chromosomes cross over. Förster Resonance Energy Transfer (FRET), where the 5'-ends of DNA strands are labeled with fluorophores, is used to determine the conformational changes of the 4WJ when interacting with DNA-binding proteins. These measurements have shown that the 4WJ opens upon binding with IHF, which is consistent with data promoting the protein's involvement in recombinase action. Subsequent FRET experiments performed confirm this observation, and provide a value for the efficiency of binding and the degree of distortion induced. Interactions between IHF and the cos I1 site of DNA from the lambda bacteriophage will also be explored. It is known that IHF induces bending at cos sites. The affinity of binding to Δ cos DNA is compared to that between IHF and its consensus sequence (H1) in E. coli DNA. Gel mobility shift assay (GMSA) and solutions experiments will measure the binding and affinity of IHF with the cos I1 site. FRET experiments with labeled cosI1 DNA will reveal the distortion induced upon IHF binding and these results will be compared to those with IHF binding to the H1 site.

Conformational Dynamics in Designer Cellulosomes Studied by Single-Pair Fret with Mfd-Pie

Cellulose is the most abundant biopolymer on earth and when broken down into oligosugars, it can be used for the production of bioethanol. As such, cellulose holds great potential as a novel energy source. The anaerobic thermophilic bacterium Clostridium thermocellum expresses an extracellular multi-enzyme complex that degrades cellulosose: the cellulosome. Cellulosomes, in general, consist of a scaffoldin domain harboring binding sites for multiple cellulases. These enzymes interact via their dockerin domain with complementary cohesin domain on the scaffoldin. An emerging research field aims at engineering ‘designer cellulosomes’ by genetically coupling different types of cohesins. These novel minimal cellulosomes should provide similar, if not better, enzymatic activity as the wild-type. Little is known about the exact role of the linker peptides between cohesin moieties. To address this, we have site-specifically labeled a designer cellulosome consisting of two cohesin subunits connected by either a natural or shortened peptide linker and performed single pair Forster resonance energy transfer experiments using pulsed interleaved excitation and multi-parameter fluorescence detection. We observe different conformational states of the cohesin dimer, suggesting the presence of conformational dynamics. Relating the conformational dynamics of such designer cellulosomes with their activity will be of great help for understanding and improving their function.

Preparation and characterization of anionic oligopeptide-modified tissue plasminogen activator for triggered delivery: An approach for localized thrombolysis

PurposeThe study sought to synthesize anionic peptide-conjugated tissue plasminogen activator (tPA) for its targeted/triggered delivery, where tPA's activity would be masked in the circulation and regenerated at the thrombus site by a commonly used anticoagulant, heparin, to minimize tPA associated bleeding complications. MethodstPA was conjugated to Polyglutamate, and the activity of oligoanion-modified tPA was tested by fibrinolytic assay. Separately human serum albumin (HSA) was conjugated to protamine and the formation of its electrostatic complex with anionic peptide was monitored by Förster Resonance Energy Transfer (FRET). The masking of tPA-activity via steric hindrance created by albumin, and subsequent regeneration with therapeutic dose of heparin was tested by enzymatic assay. Stability of ‘camouflaged-tPA’ was determined in human plasma. Using fluorescence microscope, binding of camouflaged-tPA with activated platelets was monitored. Heparin modulated clot-lysis was evaluated in human blood clot. ResultsThe anionic tPA retained ~97% activity of the unmodified-tPA. FRET experiments confirmed the electrostatic interaction between polyglutamate and protamine which was subsequently reversed by heparin. Complexation with HSA-protamine masked ~60% of tPA activity which was fully regenerated by heparin. The complex retained its prodrug character in human plasma after incubation at 37°C. Fluorescence microscopic study confirmed binding of the construct with activated platelets. In lysing human clot, the camouflage could mask tPA-activity until it was triggered at a heparin level of 0.4U/mL. ConclusionOligoanion-modified tPA could be used for targeted/triggered delivery where its enzymatic activity could be masked by HSA-protamine conjugate and successfully regenerated by therapeutic dose of heparin.

Förster Resonance Energy Transfer (FRET) between Heterogeneously Distributed Probes: Application to Lipid Nanodomains and Pores

The formation of membrane heterogeneities, e.g., lipid domains and pores, leads to a redistribution of donor (D) and acceptor (A) molecules according to their affinity to the structures formed and the remaining bilayer. If such changes sufficiently influence the Förster resonance energy transfer (FRET) efficiency, these changes can be further analyzed in terms of nanodomain/pore size. This paper is a continuation of previous work on this theme. In particular, it is demonstrated how FRET experiments should be planned and how data should be analyzed in order to achieve the best possible resolution. The limiting resolution of domains and pores are discussed simultaneously, in order to enable direct comparison. It appears that choice of suitable donor/acceptor pairs is the most crucial step in the design of experiments. For instance, it is recommended to use DA pairs, which exhibit an increased affinity to pores (i.e., partition coefficients KD,A > 10) for the determination of pore sizes with radii comparable to the Förster radius R0. On the other hand, donors and acceptors exhibiting a high affinity to different phases are better suited for the determination of domain sizes. The experimental setup where donors and acceptors are excluded from the domains/pores should be avoided.

Simple Estimation of Förster Resonance Energy Transfer (FRET) Orientation Factor Distribution in Membranes

Because of its acute sensitivity to distance in the nanometer scale, Förster resonance energy transfer (FRET) has found a large variety of applications in many fields of chemistry, physics, and biology. One important issue regarding the correct usage of FRET is its dependence on the donor-acceptor relative orientation, expressed as the orientation factor κ2. Different donor/acceptor conformations can lead to κ2 values in the 0 ≤ κ2 ≤ 4 range. Because the characteristic distance for FRET, R0, is proportional to (κ2)1/6, uncertainties in the orientation factor are reflected in the quality of information that can be retrieved from a FRET experiment. In most cases, the average value of κ2 corresponding to the dynamic isotropic limit (<κ2> = 2/3) is used for computation of R0 and hence donor-acceptor distances and acceptor concentrations. However, this can lead to significant error in unfavorable cases. This issue is more critical in membrane systems, because of their intrinsically anisotropic nature and their reduced fluidity in comparison to most common solvents. Here, a simple numerical simulation method for estimation of the probability density function of κ2 for membrane-embedded donor and acceptor fluorophores in the dynamic regime is presented. In the simplest form, the proposed procedure uses as input the most probable orientations of the donor and acceptor transition dipoles, obtained by experimental (including linear dichroism) or theoretical (such as molecular dynamics simulation) techniques. Optionally, information about the widths of the donor and/or acceptor angular distributions may be incorporated. The methodology is illustrated for special limiting cases and common membrane FRET pairs.

Lateral distribution of NBD-PC fluorescent lipid analogs in membranes probed by molecular dynamics-assisted analysis of Förster Resonance Energy Transfer (FRET) and fluorescence quenching.

Förster resonance energy transfer (FRET) is a powerful tool used for many problems in membrane biophysics, including characterization of the lateral distribution of lipid components and other species of interest. However, quantitative analysis of FRET data with a topological model requires adequate choices for the values of several input parameters, some of which are difficult to obtain experimentally in an independent manner. For this purpose, atomistic molecular dynamics (MD) simulations can be potentially useful as they provide direct detailed information on transverse probe localization, relative probe orientation, and membrane surface area, all of which are required for analysis of FRET data. This is illustrated here for the FRET pairs involving 1,6-diphenylhexatriene (DPH) as donor and either 1-palmitoyl,2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)- sn-glycero-3-phosphocholine (C6-NBD-PC) or 1-palmitoyl,2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphocholine (C12-NBD-PC) as acceptors, in fluid vesicles of 1,2-dipalmitoyl-sn-3-glycerophosphocholine (DPPC, 50 °C). Incorporation of results from MD simulations improves the statistical quality of model fitting to the experimental FRET data. Furthermore, the decay of DPH in the presence of moderate amounts of C12-NBD-PC (>0.4 mol%) is consistent with non-random lateral distribution of the latter, at variance with C6-NBD-PC, for which aggregation is ruled out up to 2.5 mol% concentration. These conclusions are supported by analysis of NBD-PC fluorescence self-quenching. Implications regarding the relative utility of these probes in membrane studies are discussed.

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium, cAMP, inositol phosphates and cGMP. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker.