Forster Resonance Energy Transfer Experiments Research Articles

Microwave analogy of Förster resonance energy transfer and effect of finite antenna length

The near-field interaction between quantum emitters, governed by Förster resonance energy transfer (FRET), plays a pivotal role in nanoscale energy transfer mechanisms. However, FRET measurements in the optical regime are challenging as they require nanoscale control of the position and orientation of the emitters. To overcome these challenges, microwave measurements were proposed for enhanced spatial resolution and precise orientation control. However, unlike in optical systems for which the dipole can be taken to be infinitesimal in size, the finite size of microwave antennas can affect energy transfer measurements, especially at short distances. This highlights the necessity to consider the finite antenna length to obtain accurate results. In this study, we advance the understanding of dipole–dipole energy transfer in the microwave regime by developing an analytical model that explicitly considers finite antennas. Unlike previous works, our model calculates the mutual impedance of finite-length thin-wire dipole antennas without assuming a uniform current distribution. We validate our analytical model through experiments investigating energy transfer between antennas placed adjacent to a perfect electric conductor mirror. This allows us to provide clear guidelines for designing microwave experiments, distinguishing conditions where finite-size effects can be neglected and where they must be taken into account. Our study not only contributes to the fundamental physics of energy transfer but also opens avenues for microwave antenna impedance-based measurements to complement optical FRET experiments and quantitatively explore dipole–dipole energy transfer in a wider range of conditions.

Influence of the Fluorophore Mobility on Distance Measurements by Gas-Phase FRET.

Gas-phase Förster resonance energy transfer (FRET) combines mass spectrometry and fluorescence spectroscopy for the conformational analysis of mass-selected biomolecular ions. In FRET, fluorophore pairs are typically covalently attached to a biomolecule using short linkers, which affect the mobility of the dye and the relative orientation of the transition dipole moments of the donor and acceptor. Intramolecular interactions may further influence the range of motion. Yet, little is known about this factor, despite the importance of intramolecular interactions in the absence of a solvent. In this study, we applied transition metal ion FRET (tmFRET) to probe the mobility of a single chromophore pair (Rhodamine 110 and Cu2+) as a function of linker lengths to assess the relevance of intramolecular interactions. Increasing FRET efficiencies were observed with increasing linker length, ranging from 5% (2 atoms) to 28% (13 atoms). To rationalize this trend, we profiled the conformational landscape of each model system using molecular dynamics (MD) simulations. We captured intramolecular interactions that promote a population shift toward smaller donor-acceptor separation for longer linker lengths and induce a significant increase in the acceptor's transition dipole moment. The presented methodology is a first step toward the explicit consideration of a fluorophore's range of motion in the interpretation of gas-phase FRET experiments.

FRETing about the details: Case studies in the use of a genetically encoded fluorescent amino acid for distance-dependent energy transfer.

Förster resonance energy transfer (FRET) is a valuable method for monitoring protein conformation and biomolecular interactions. Intrinsically fluorescent amino acids that can be genetically encoded, such as acridonylalanine (Acd), are particularly useful for FRET studies. However, quantitative interpretation of FRET data to derive distance information requires careful use of controls and consideration of photophysical effects. Here we present two case studies illustrating how Acd can be used in FRET experiments to study small molecule induced conformational changes and multicomponent biomolecular complexes.

Resolution of Maximum Entropy Method-Derived Posterior Conformational Ensembles of a Flexible System Probed by FRET and Molecular Dynamics Simulations

Maximum entropy methods (MEMs) determine posterior distributions by combining experimental data with prior information. MEMs are frequently used to reconstruct conformational ensembles of molecular systems for experimental information and initial molecular ensembles. We performed time-resolved Förster resonance energy transfer (FRET) experiments to probe the interdye distance distributions of the lipase-specific foldase Lif in the apo state, which likely has highly flexible, disordered, and/or ordered structural elements. Distance distributions estimated from ensembles of molecular dynamics (MD) simulations serve as prior information, and FRET experiments, analyzed within a Bayesian framework to recover distance distributions, are used for optimization. We tested priors obtained by MD with different force fields (FFs) tailored to ordered (FF99SB, FF14SB, and FF19SB) and disordered proteins (IDPSFF and FF99SBdisp). We obtained five substantially different posterior ensembles. As in our FRET experiments the noise is characterized by photon counting statistics, for a validated dye model, MEM can quantify consistencies between experiment and prior or posterior ensembles. However, posterior populations of conformations are uncorrelated to structural similarities for individual structures selected from different prior ensembles. Therefore, we assessed MEM simulating varying priors in synthetic experiments with known target ensembles. We found that (i) the prior and experimental information must be carefully balanced for optimal posterior ensembles to minimize perturbations of populations by overfitting and (ii) only ensemble-integrated quantities like inter-residue distance distributions or density maps can be reliably obtained but not ensembles of atomistic structures. This is because MEM optimizes ensembles but not individual structures. This result for a highly flexible system suggests that structurally varying priors calculated from varying prior ensembles, e.g., generated with different FFs, may serve as an ad hoc estimate for MEM reconstruction robustness.

Evaluation by Experimentation and Simulation of a FRET Pair Comprising Fluorescent Nucleobase Analogs in Nucleosomes.

Förster resonance energy transfer (FRET) is an attractive tool for understanding biomolecular dynamics. FRET-based analysis of nucleosomes has the potential to fill the knowledge gaps between static structures and dynamic cellular behaviors. Compared with typical FRET pairs using bulky fluorophores introduced by flexible linkers, fluorescent nucleoside-based FRET pair has great potential since it can be fitted within the helical structures of nucleic acids. Herein we report on the construction of nucleosomes containing a nucleobase FRET pair and the investigation of experimental and theoretical FRET efficiencies through steady-state fluorescence spectroscopy and calculation based on molecular dynamics simulations, respectively. Distinguishable experimental FRET efficiencies were observed depending on the positions of FRET pairs in nucleosomal DNA. The tendency could be supported by theoretical study. This work suggests the possibility of our approach to analyze structural changes of nucleosomes by epigenetic modifications or internucleosomal interactions.

Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for FRET measurements.

Förster resonance energy transfer (FRET) is a radiationless interaction between a donor and an acceptor whose distance dependence makes it a sensitive tool for studying the oligomerization and the structure of proteins. When FRET is determined by measuring the sensitized emission of the acceptor, a parameter characterizing the ratio of detection efficiencies of an excited acceptor versus an excited donor is invariably involved in the formalism. For FRET measurements involving fluorescent antibodies or other external labels, this parameter, designated by α, is usually determined by comparing the intensity of a known number of donors and acceptors in two independent samples leading to a large statistical variability if the sample size is small. Here, we present a method that improves precision by applying microbeads with a calibrated number of antibody binding sites and a donor-acceptor mixture in which donors and acceptors are present in a certain, experimentally determined ratio. A formalism is developed for determining α and the superior reproducibility of the proposed method compared to the conventional approach is demonstrated. Since the novel methodology does not require sophisticated calibration samples or special instrumentation, it can be widely applied for the quantification of FRET experiments in biological research.

Shrinking gate fluorescence correlation spectroscopy yields equilibrium constants and separates photophysics from structural dynamics

Fluorescence correlation spectroscopy is a versatile tool for studying fast conformational changes of biomolecules especially when combined with Förster resonance energy transfer (FRET). Despite the many methods available for identifying structural dynamics in FRET experiments, the determination of the forward and backward transition rate constants and thereby also the equilibrium constant is difficult when two intensity levels are involved. Here, we combine intensity correlation analysis with fluorescence lifetime information by including only a subset of photons in the autocorrelation analysis based on their arrival time with respect to the excitation pulse (microtime). By fitting the correlation amplitude as a function of microtime gate, the transition rate constants from two fluorescence-intensity level systems and the corresponding equilibrium constants are obtained. This shrinking-gate fluorescence correlation spectroscopy (sg-FCS) approach is demonstrated using simulations and with a DNA origami-based model system in experiments on immobilized and freely diffusing molecules. We further show that sg-FCS can distinguish photophysics from dynamic intensity changes even if a dark quencher, in this case graphene, is involved. Finally, we unravel the mechanism of a FRET-based membrane charge sensor indicating the broad potential of the method. With sg-FCS, we present an algorithm that does not require prior knowledge and is therefore easily implemented when an autocorrelation analysis is carried out on time-correlated single-photon data.

Gas-phase Förster resonance energy transfer in mass-selected and trapped ions.

Förster Resonance Energy transfer (FRET) is a nonradiative process that may occur from an electronically excited donor to an acceptor when the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor. FRET experiments have been done in the gas phase based on specially designed mass-spectroscopy setups with the goal to obtain structural information on biomolecular ions labeled with a FRET pair (i.e., donor and acceptor dyes) and to shed light on the energy-transfer process itself. Ions are accumulated in a radio-frequency ion trap or a Penning trap where mass selection of those of interest takes place, followed by photoexcitation. Gas-phase FRET is identified from detection of emitted light either from the donor, the acceptor, or both, or from a fragmentation channel that is specific to the acceptor when electronically excited. The challenge associated with the first approach is the collection and detection of photons emitted from a thin ion cloud that is not easily accessible while the second approach relies both on the photophysical and chemical behavior of the acceptor. In this review, we present the different instrumentation used for gas-phase FRET, including a discussion of advantages and disadvantages, and examples on how the technique has provided important structural information that is not easily obtainable otherwise. Furthermore, we describe how the spectroscopic properties of the dyes are affected by nearby electric fields, which is readily discernable from experiments on simple model systems with alkyl or π-conjugated bridges. Such spectral changes can have a significant effect on the FRET efficiency. Ideas for new directions are presented at the end with special focus on cold-ion spectroscopy.

Fast Screening of Protein-Protein Interactions Using Förster Resonance Energy Transfer (FRET-) Based Fluorescence Plate Reader Assay in Live Cells.

Protein-protein interactions (PPIs) have great importance for intracellular signal transduction and sustaining the homeostasis of an organism. Thus, the identification of PPIs is necessary to better understand the downstream signaling functions of the proteins in healthy and pathological conditions. Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool for detecting PPIs in living cells. In literature, FRET analysis methods such as donor photobleaching (FLIM), acceptor photobleaching, spectral imaging, and the three-filter cube method (sensitized emission) are abundantly applied to investigate PPIs; however, they require various expensive instrumentations, and their calculation methods are very time consuming. Since confocal microscopy applications and live cell-based techniques of FRET are very costly, scientists sometimes prefer plate readers for FRET experiments. However, plate reader applications also have many disadvantages and considerations compared to confocal fluorescence microscopy, and complex calculation procedures should be performed. To overcome these problems, we propose a FRET-based high-throughput assay method with a standard monochromator-based microplate reader, which is generally available in most biochemistry laboratories, and an alternative calculation procedure. This rapid, low cost, and effective analysis method enables the scientists to prescreen PPIs in living cells as a preliminary study and quick glance at the experiment before preparing the whole experimental setup with the expensive instrumentations. Additionally, the alternative calculation procedure provides the FRET area comparison without complex bleed-through calculations in a non-conventional manner by shortening the analysis processes with this quick and uncomplicated spectral representation.

Key Steps to Follow in a FRET Experiment

Förster resonance energy transfer (FRET) is a versatile part of the toolbox of fluorescence methods. This through-space, photon-less energy transfer process between a donor fluorophore and an acceptor chromophore is perhaps most famous for its utility as a “molecular ruler” that can resolve nanometer-scale distances. FRET is also a popular and advantageous basis for biomolecular assays and sensors.

Unraveling multi-state molecular dynamics in single-molecule FRET experiments. II. Quantitative analysis of multi-state kinetic networks.

Single-molecule Förster Resonance Energy Transfer (smFRET) experiments are ideally suited to resolve the structural dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, mainly when these dynamics occur on the sub-millisecond timescale. Many methods for quantitative analysis are challenged if more than two states are involved, and the appropriate choice of the number of states in the kinetic network is difficult. An additional complication arises if dynamically active molecules coexist with pseudo-static molecules in similar conformational states with undistinguishable Förster Resonance Energy Transfer (FRET) efficiencies. To address these problems, we developed a quantitative integrative analysis framework that combines the information from FRET-lines that relate average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms, fluorescence decays obtained by time-correlated single-photon-counting, photon distribution analysis of the intensities, and fluorescence correlation spectroscopy. Individually, these methodologies provide ambiguous results for the characterization of dynamics in complex kinetic networks. However, the global analysis approach enables accurate determination of the number of states, their kinetic connectivity, the transition rate constants, and species fractions. To challenge the potential of smFRET experiments for studying multi-state kinetic networks, we apply our integrative framework using a set of synthetic data for three-state systems with different kinetic connectivity and exchange rates. Our methodology paves the way toward an integrated analysis of multiparameter smFRET experiments that spans all dimensions of the experimental data. Finally, we propose a workflow for the analysis and show examples that demonstrate the usefulness of this toolkit for dynamic structural biology.

Recent Efforts and Milestones for Simulating Nucleic Acid FRET Experiments through Computational Methods.

Computational methods can greatly aid nucleic acid fluorescence experiments by either offering fully detailed atomic insights into the conformations and interactions present in the studied system or by providing accurate simulations of the fundamental parameters. Fluorescence-based optical biosensors show great potential for clinical diagnosis of life-altering diseases with a very high specificity. Many of the designs for such rely on the concept of Förster resonance energy transfer (FRET). Currently, the methods used experimentally make use of theoretical assumptions which fundamentally affect the results. Having a detailed atomistic overview or significant simulated parameters could improve the understanding of the calculations and provide much more accurate outcomes. However, there are many challenges that need to be addressed before standardized computational protocols can be employed. This review is meant to highlight the progress made for computational methods used to simulate FRET experiments for nucleic acid probes. Recent advances have been made in computational tools, such as force field parametrizations and improved protocols. Complementary simulations to experimental data are also comprised in the this review.

Analysis of the conformational space and dynamics of RNA helicases by single-molecule FRET in solution and on surfaces.

RNA helicases are a diverse group of enzymes that catalyze the unwinding of RNA duplex regions in an ATP-dependent reaction. Both the helicase itself and its RNA substrate undergo conformational changes during the reaction, which are amenable to Förster resonance energy transfer (FRET) studies. Single-molecule FRET studies in solution by confocal microscopy and on surfaces by total internal reflection microscopy provide information on different conformers present, their fractional populations in equilibrium, and the rate constants of their inter-conversion. Collectively, the information gained can be integrated into a kinetic and thermodynamic framework that quantitatively describes the conformational dynamics of the helicase studied. FRET experiments also provide distance information to map and model the structures of individual conformational states. The integrated model provides a comprehensive description of the structure and dynamics of the helicase, which can be linked to its biological function. Single-molecule FRET studies have tremendous potential to define the relationship between structure, function and dynamics of RNA helicases and to understand the mechanistic basis for their broad range of biological functions. The focus of this chapter is on providing guidance in the design of single-molecule FRET experiments and on the interpretation of the data obtained. Selected examples illustrate important considerations when analyzing single-molecule experiments, as well as their limitations and possible pitfalls.

Chemical Dual End-Labeling of Large Ribozymes.

Fast and efficient site-specific labeling of long RNAs is one of the main bottlenecks limiting distance measurements by means of Förster resonance energy transfer (FRET) or electron paramagnetic resonance (EPR) spectroscopy. Here, we present an optimized protocol for dual end-labeling with different fluorophores at the same time meeting the restrictions of highly labile and degradation-sensitive RNAs. We describe in detail the dual-labeling of a catalytically active wild-type group II intron as a typical representative of long functional RNAs. The modular procedure chemically activates the 5'-phosphate and the 3'-ribose for bioconjugation with a pair of fluorophores, as shown herein, or with spin labels. The mild reaction conditions preserve the structural and functional integrity of the biomacromolecule and results in covalent, dual-labeled RNA in its pre-catalytic state in yields suitable for both ensemble and single-molecule FRET experiments.

TdLanYFP, a Yellow, Bright, Photostable, and pH-Insensitive Fluorescent Protein for Live-Cell Imaging and Förster Resonance Energy Transfer-Based Sensing Strategies.

Yellow fluorescent proteins (YFPs) are widely used as optical reporters in Förster resonance energy transfer (FRET)-based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pH values. Here, we characterize the yellow fluorescent protein tdLanYFP, derived from the tetrameric protein from the cephalochordate Branchiostoma lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133,000 mol-1·L·cm-1, it is, to our knowledge, the brightest dimeric fluorescent protein available. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and in live cells. As a consequence, tdLanYFP allows imaging of cellular structures with subdiffraction resolution using STED nanoscopy and is compatible with the use of spectromicroscopies in single-molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pH values. Finally, we show that tdLanYFP is a valuable FRET partner either as a donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFP a very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging including FRET experiments at acidic pH.

Förster Resonance Energy Transfer Measurements in Living Plant Cells.

Sensitized emission-based Förster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

Elucidation of Interactions Between Integration Host Factor and A DNA Four‐Way Junction

Integration host factor (IHF) is an E. coli nucleoid-associated protein that was initially discovered in the integration of bacterial phage DNA. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence and induces a bend of ~160° upon binding. Our previous results have shown that IHF binds to DNA 4WJ that do not contain the consensus sequence with high affinity. DNA Four-Way Junctions (4WJ) are important intermediates in double-strand break repair and homologous recombination. These junctions can adopt either an open or stacked conformation, in which the open conformation facilitates the process of branch migration and strand exchange. In this study, we investigated the IHF-induced changes in the population distribution of junction conformations, using ensemble and single molecule Förster resonance energy transfer (FRET) methods. Our steady state and time-resolved FRET data indicated that under conditions stabilizing the stacked junction conformation, upon binding, IHF induces the open conformation of junction. Single-molecule FRET (smFRET) experiments, performed with total internal reflection fluorescence microscopy, measured population distributions of the junction and showed clear opening of the junctions upon IHF binding. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Cross correlation analyses reveal that the protein slows down the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+, consistent with stabilization of the open conformation. These findings which show that IHF binds to 4WJ with high affinity, induces the open conformation and alters conformational dynamics, suggest that IHF may play multiple roles in the processes of integration and recombination.

The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility.

Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor’s direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY‐binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.

Development and characterization of layer-by-layer coated liposomes with poly(L-lysine) and poly(L-glutamic acid) to increase their resistance in biological media.

Multilayered coated liposomes were prepared using the layer-by-layer (LbL) technique in an effort to improve their stability in biological media. The formulation strategy was based on the alternate deposition of two biocompatible and biodegradable polyelectrolytes - poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) - on negatively charged small unilamellar vesicles (SUVs). Some parameters of the formulation process were optimized such as the polyelectrolyte concentration and the purification procedure. This optimized procedure has allowed the development of very homogeneous formulations of liposomes coated with up to 6 layers of polymers (so-called layersomes). The coating was characterized by dynamic light scattering (DLS), zeta potential measurements and Förster resonance energy transfer (FRET) between two fluorescently labeled polyelectrolytes. Studies on the stability of the formulations at 4°C in a buffered solution have shown that most structures are stable over 1month without impacting their encapsulation capacity. In addition, fluorophore release experiments have demonstrated a better resistance of the layersomes in the presence of a non-ionic detergent (Triton™ X-100) as well as in the presence of phospholipase A2 and human plasma. In conclusion, new multilayered liposomes have been developed to increase the stability of conventional liposomes in biological environments.

Distinct Conformational States Underlie Pausing during Initiation of HIV-1 Reverse Transcription

A hallmark of the initiation step of HIV-1 reverse transcription, in which viral RNA genome is converted into double-stranded DNA, is that it is slow and non-processive. Biochemical studies have identified specific sites along the viral RNA genomic template in which reverse transcriptase (RT) stalls. These stalling points, which occur after the addition of three and five template dNTPs, may serve as checkpoints to regulate the precise timing of HIV-1 reverse transcription following viral entry. Structural studies of reverse transcriptase initiation complexes (RTICs) have revealed unique conformations that may explain the slow rate of incorporation; however, questions remain about the temporal evolution of the complex and features that contribute to strong pausing during initiation. Here we present cryo-electron microscopy and single-molecule characterization of an RTIC after three rounds of dNTP incorporation (+3), the first major pausing point during reverse transcription initiation. Cryo-electron microscopy structures of a +3 extended RTIC reveal conformational heterogeneity within the RTIC core. Three distinct conformations were identified, two of which adopt unique, likely off-pathway, intermediates in the canonical polymerization cycle. Single-molecule Förster resonance energy transfer experiments confirm that the +3 RTIC is more structurally dynamic than earlier-stage RTICs. These alternative conformations were selectively disrupted through structure-guided point mutations to shift single-molecule Förster resonance energy transfer populations back toward the on-pathway conformation. Our results support the hypothesis that conformational heterogeneity within the HIV-1 RTIC during pausing serves as an additional means of regulating HIV-1 replication.