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TECHNIQUES

Fluorescence resonance energy transfer (FRET)

by Hays Rye

Overview — The basics
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Purpose

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Fluorescence resonance energy transfer (FRET or RET) is a technique that monitors the distance between different fluorescent probes that are attached to macromolecules. FRET can be employed to monitor either binding interactions between different molecules or conformational changes within the same molecule. This technique can be used both in vitro and in vivo.

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The concept

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Figure 1  
Excitation and emission involves transitions between the ground state and the excited state of a fluorophore. Upon excitation with a photon of light (hν1), the electronic structure of the fluorophore rapidly rearranges into one of a number of energetically similar states that decay to the lowest energy excited state. Following a delay (anywhere from a fraction of a nanosecond to many milliseconds, depending upon the molecular structure of the fluorophore), the excited state decays back to the ground state with the emission of a photon of light of lower energy (hν2). This process is observable in the absorption and emission spectra of the fluorescent probe.

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The changes that occur when a fluorescent probe (a fluorophore) absorbs and emits light are at the heart of FRET (4861; 4860). The absorption of a photon of light by a fluorophore induces a rearrangement of the electronic structure of the molecule, briefly storing some of the energy of the absorbed light in an excited state Figure 1). After a short period of time (anywhere from a few picoseconds to a few milliseconds, depending on the fluorophore), the electronic structure of the probe relaxes back to its equilibrium state (the ground state). The relaxation of the excited state to its associated ground state occurs with the emission of a photon of fluorescent light at a lower energy (longer wavelength) than the light originally absorbed.

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Figure 2  
FRET involves the transfer of energy from the excited state of the donor to the excited state of the acceptor. The altered charge distribution of the donor can drive the acceptor into its excited state if the two states are correctly matched and the molecules are close in space. This results in the transfer of energy from the donor to the acceptor and the relaxation of the donor to its ground state. Note that this process is a direct energetic coupling between the electronic states of the two fluorophores and is not due to the emission of a photon of light from the donor and its absorption by the acceptor. If the acceptor is fluorescent, it can then emit a photon of light at its characteristic emission wavelengths.

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FRET is essentially a two-step version of the same excitation and emission process, with a slight wrinkle. The energy stored by the initially excited fluorescent probe (the "donor") can be transferred to another fluorescent probe (the "acceptor") under certain conditions. Essentially, the perturbed charge distribution of the excited donor can influence the electronic structure of the acceptor and drive the acceptor into an excited state. This results in the relaxation of the donor and the "radiationless" transfer of energy from the donor to the acceptor without the emission of a photon from the donor (Figure 2). If the acceptor probe is fluorescent, it will then go on to relax to its ground state with the emission of a photon of light, generally at much longer wavelengths than the donor can emit itself. Thus, transfer is detected when a sample irradiated with light of a wavelength that only the donor can absorb emits at a wavelength only possible from the acceptor.

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Figure 3  
FRET can be used to measure the distance between two different parts of a macromolecule. Three modified versions of the molecule are prepared: (1) with donor-only, (2) with acceptor-only, and (3) with donor plus acceptor. The fluorescence emission spectra of the donor-only and acceptor-only molecules (excited at λ1) are shown. Note that the acceptor-only sample shows little fluorescence when excited at λ1, since it has low absorbance at this wavelength. If the donor and acceptor groups are close enough together, when a molecule modified with both is excited at the donor excitation wavelength the emission spectrum shows characteristic quenching of the donor emission and enhancement of the acceptor emission. Quantitation of the amount of quenching or enhancement can be used to calculate the distance between the labeled points on the macromolecule.

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Figure 4  
The efficiency with which energy is transferred between two flourophores depends on the distance between them. Beyond a certain distance, little transfer occurs and the emission spectrum is dominated by the properties of the donor (top). When the flourophores are closer together, FRET can occur and the emission spectrum is dominated by the acceptor (4859).

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In a typical FRET experiment, a sample is first prepared with donor and acceptor fluorophores attached to biomolecules of interest. The sample is then excited at a wavelength of light that is optimally absorbed by the donor but not by the acceptor. Energy transfer is then detected as a decrease (or quenching) in the donor fluorescence and an increase (or enhancement) in the acceptor fluorescence, in comparison to control experiments with each of the flourophores alone (Figure 3). Whether or not energy transfer occurs depends on a number of physical parameters, perhaps the most useful being the distance between the donor and acceptor probes (Figure 4) (4848). Because of this, probes positioned on different macromolecules can be used to monitor changes in their proximity, while probes attached at different points on the same molecule can be used to detect movement between its parts.

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As a method to investigate molecular distances and changes in molecular proximity, FRET is generally applicable to most biological systems. The basic requirements for a FRET experiment are: (1) a fluorescence detection system and (2) a method to attach appropriately matched fluorescent probes to the biomolecules of interest. Most standard fluorescence instruments can be adapted for FRET experiments, including fluorescence spectrometers and fluorescence microscopes. Fluorescent probes suitable for FRET can range from intrinsic probes (naturally bound cofactors, tryptophan residues in proteins, etc.), to fusion proteins (e.g., green fluorescent protein [GFP] and its cousins of other colors), to chemically attached organic molecules or organometallic complexes.

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Details and variations

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Figure 5  
FRET between two fluorescent probes requires that a significant overlap exists between the donor emission spectrum and the acceptor absorption spectrum.

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FRET depends on a number of factors and conditions (4861; 4860; 4849; 4850). One essential requirement is overlap between the donor emission spectrum and the acceptor absorption spectrum. If there is not sufficient overlap, the donor will not couple to the acceptor and FRET cannot occur (Figure 5). Additional physical characteristics that govern FRET include: the quantum yield of the donor (the fraction of absorbed photons it reemits as fluorescent photons), the extinction coefficient of the acceptor (how efficiently it absorbs photons at its excitation wavelength), and the relative spatial orientation of the donor and acceptor probes. These parameters, together with the distance between the donor and acceptor, determine how efficiently energy is transferred from the donor to the acceptor.

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Under a given set of conditions, each pair of FRET probes can be characterized by a parameter known as the Förster distance, R0, which is the distance at which 50% of the excitation energy is transferred from the donor to the acceptor. In the simplest application of FRET, the magnitude of donor quenching can be combined with the value of R0 to calculate the distance between the probes in the following manner:

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By measuring the donor fluorescence intensity in the absence (ID) and presence of the acceptor (IDA), one can calculate the energy transfer efficiency (E):

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The transfer efficiency is then directly related to the distance (r) between the donor and acceptor by:

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It is also possible to use the enhancement of the acceptor fluorescence rather than the quenching of the donor fluorescence for calculating the separation of the probes, but this requires some modifications to these equations and adds additional technical complexity to the measurements (4860; 4851).

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In the most favorable cases, FRET can be used to accurately measure distances between 15 and 100 Å (4848; 4849). This corresponds to a convenient range for molecular measurements with biological molecules, spanning distances from the width of a phospholipid bilayer (~15-20 Å), to the distance between the heme groups in hemoglobin (~25-40 Å), to the separation of two sites by 30 base pairs of double-stranded DNA (~100 Å). Note that the dependence of the transfer efficiency on the sixth-power of the distance between the probes makes FRET measurements most sensitive to the distance between the donor and acceptor when this distance is close to the value of R0 (within R0 ± 1/2 R0). So, in the design of a FRET experiment, the choice of a pair of probes depends very much on the range of distances one expects to find in the system under study. In other words, any given pair of FRET probes is useful only in a limited range of distances centered on the value of R0 for that pair.

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The accuracy of a FRET distance measurement calculated from the change in donor or acceptor fluorescence intensity depends upon: (1) the precision with which R0 has been determined, (2) how accurately the protein concentrations are known, and (3) the precision of the fluorescence intensity measurement. Alternately, the FRET distance can be calculated using the change in the donor fluorescence lifetime, an approach that eliminates the need to know the protein concentrations with great precision. This approach is more complex and requires more sophisticated instrumentation, however. A wide literature is available on the theory, procedural details, and variations on making distance measurements by FRET (4861; 4860; 4849; 4850; 4851).

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Using FRET as a proximity sensor

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Many experiments do not require the measurement of a precise distance, and a simple readout of whether two molecules are bound to (or very near) each other is all that is needed. Under these conditions, FRET can be used as a powerful and sensitive method for studying changes in the proximity of different molecules (4852). This application of FRET can be either qualitative (simply seeing if certain molecules are close to one another) or quantitative (measuring binding equilibria or kinetics), and does not require knowledge of the exact value of R0 for the probes in use.

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Figure 6  
The binding of two macromolecules to one another can be measured by FRET. A donor probe is attached to one of the two macromolecules and an acceptor probe is attached to the other. Upon mixing, binding brings the fluorescent probes close together and FRET can occur. By comparing the changes in fluorescence of donor-only, acceptor-only, and donor-plus-acceptor samples, the kinetics of binding can be measured.

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The simple assumption for this application of FRET is: when the molecules do not interact with each other, there is no energy transfer (Figure 6). At the concentrations used for most biological experiments (well below millimolar), this condition is generally met. Only when the pair of molecules bind to one another are the probes brought close enough together for energy transfer to take place.

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Advantages of FRET

  • Extremely high sensitivity permits measurements to be made on very small quantities of material and in very small volumes. Ultimate sensitivity is limited primarily by the instrumentation available.
  • Can be applied to any molecular system for which a fluorescent derivative can be produced.
  • Measures molecular distances (15-100 Å) that are similar to the dimensions of many biological macromolecules and are difficult to study with other methods.
  • Is capable of providing both proximity and kinetic information over a wide range of time scales (nanoseconds to hours).
  • Through the use of fluorescent proteins like GFP, FRET can be readily applied to the study of molecular interactions inside living cells.
  • Easily adapted to a wide variety of instrument formats.

Controls, considerations, and disadvantages

  • Accurate distance measurement is generally limited to a single pair of sites in any given experiment.
  • Various physical changes in the molecular system (e.g., probe microenvironment) can alter the fluorescence properties of the probes. These changes can mimic FRET and must be controlled for very carefully. If accurate distance measurements are required, time-consuming and complex additional analysis can be necessary. Even simple proximity studies can require that several measurements be made to ensure the changes seen are due to FRET and not other trivial modifications of a probe's fluorescence properties.
  • The requirement for the attachment of fluorescent probes to a molecule of interest often requires the creation of fusion proteins, mutation and/or chemical modification of the molecules under study. These changes can perturb the function or characteristics of the molecules to be examined and must be controlled for.
  • Restricting the rotational mobility of fluorescent probes used for FRET can make accurate distance calculation difficult or impossible. This is an especially serious problem for FRET measurements made with GFP variants (4852). However, this does not prevent the use of GFP-fusion proteins as proximity sensors.

Applications

Figure 7  
FRET has been employed to study the conformational changes that occur in Type II myosin during the ATP-driven movement of this molecular motor. Small organic fluorophores were attached to different parts of the myosin protein. Changes in distance between these regions of the protein during the myosin power stroke can then be readily detected as changes in the efficiency of energy transfer between the fluorophores (4853). Note that only one of the two donor fluorophores was attached to myosin in each experiment.
Credits:

Courtesy of Jim Spudich. Reprinted from Shih et al., 2000 (4853), © 2000, with permission from Elsevier.

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Figure 8  
Using FRET to study the mechanism of assisted protein folding by the molecular chaperone GroEL. A donor fluorophore (AEDANS) was attached to GroEL and an acceptor (5IAF) to the cochaperonin GroES. The structures of these small organic probes are shown at their attachment sites on GroEL and GroES. The inset shows a magnified view of how the two fluorophores are positioned adjacent to one another when the GroEL-GroES complex forms. The formation and dissociation kinetics of the GroEL-GroES complex were studied by FRET (967).
Figure 9  
Myosin light chain kinase (MLCK) activates myosin and is itself activated by the binding of calmodulin. An in vivo sensor that specifically detects MLCK activity is shown at the top. Attached to MLCK are GFP and BFP (a variant of GFP that flouresces blue) connected by a short, flexible region with a calmodulin binding site. When calmodulin is bound to this site the region is held rigid, so that the GFP and BFP cannot come close enough together for energy transfer to occur. The pictures show a cell expressing this construct as the cell begins to move. Long, thin stress fibers are visible in green initially because MLCK localized in them is inactive. As the MLCK is activated, blue appears in the stress fibers and the cell visibly contracts.
Credits:

Images courtesy of Rex Chisholm.

  • Determining the distance between two sites on the same molecule: detecting conformational changes.
    For example, measuring the conformational changes that occur in Type II myosin during the ATP-driven movement of this molecular motor protein (Figure 7). For this type of experiment, small, fluorescent organic dyes are attached to different parts of the same molecule and changes in distance between the labeled domains are tracked as the molecule performs its function (4853).
  • Protein-protein and protein-ligand interactions, both in vitro and in vivo.
    For example, studying the mechanism of GroEL-assisted protein folding (Figure 8) (967). In this application, a donor probe is attached to GroEL and an acceptor probe to either the cochaperonin GroES or to a substrate protein. Changes in FRET can be used to monitor when and how fast the proteins bind to or dissociate from one another. The sequence of events that lead to ATP-dependent encapsulation and folding of a substrate protein inside the GroEL-GroES cavity can then be examined in detail.
  • Protein-nucleic acid interactions and nucleic acid hybridization.
  • Protein and nucleic acid folding.
  • Membrane dynamics, membrane-protein interactions, and membrane mixing.
    For example, measuring the rate and extent of membrane mixing during membrane fusion. For this assay, a target membrane is doped with small amounts of donor and acceptor-labeled lipids at concentrations that render the donor highly quenched. Upon fusion with a membrane containing no fluorescent lipid, the donor and acceptor lipids are diluted away from one another and the FRET signal decreases (4854).
  • Enzyme kinetics.
    For example, studying enzyme mechanisms by using FRET to follow the interaction of a labeled substrate and an enzyme (4855; 4856). Additionally, the rate of product formation for many enzymes (e.g. proteases, nucleases, etc) can be followed by FRET using model substrates where a donor and acceptor fluorophore are attached at the ends of the target substrate molecule and cleavage is scored as a loss in energy transfer as the substrate molecule is split (4857).
  • Intracellular sensors.
    For example, measuring the intracellular levels of Ca2+ or the activity of a protein kinase (4852; 4858). This can be accomplished through the design and expression of fusion proteins involving color mutants of GFP. Two fluorescent protein variants capable of FRET can be joined together by a third protein domain that contains the sensor function. Upon binding or interacting with the appropriate signal, the sensor domain changes conformation and alters the distance between fluorescent protein moieties, altering the detected FRET signal. An exammple of a sensor molecule that shows when and where myosin light chain kinase is active is shown in Figure 9. Alternatively, two different fusion proteins that only interact following a specific intracellular signal can be simultaneously expressed and their conditional interaction followed by changes in FRET. This application has the tremendous advantage that it allows fine spatial localization of a protein's activity within a living cell.

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Related techniques

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FRET measurement by excited-state lifetime analysis

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FRET between identical probe molecules (homo-transfer FRET)

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Luminescence energy transfer

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Single particle FRET

Last Revised on December 9, 2004

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reviews
  • 4848 Stryer, L. (1978).  Fluorescence energy transfer as a spectroscopic ruler.  Annu. Rev. Biochem. 47, 819-846.  PubMed   Journal
  • 4849 dos Remedios, C. G. and Moens, P. D. (1995).  Fluorescence resonance energy transfer spectroscopy is a reliable "ruler" for measuring structural changes in proteins. Dispelling the problem of the unknown orientation factor.  J. Struct. Biol. 115, 175-185.  PubMed   Journal
  • 4850 Wu, P. and Brand, L. (1994).  Resonance energy transfer: methods and applications.  Anal. Biochem. 218, 1-13.  PubMed   Journal
  • 4851 Clegg, R. M. (1995).  Fluorescence resonance energy transfer.  Curr. Opin. Biotechnol. 6, 103-110.  PubMed  
  • 4852 Miyawaki, A. (2003).  Visualization of the spatial and temporal dynamics of intracellular signaling.  Dev. Cell 4, 295-305.  PubMed   Journal
  • 4858 Tsien, R. Y. (1998).  The green fluorescent protein.  Annu. Rev. Biochem. 67, 509-544.  PubMed   Journal
  • 4860 Lakowicz, J. R. (1999).  Fluorescence anisotropy. In Principles of Fluorescence Spectroscopy Joseph R. Lakowicz, ed. (New York: Kluwer Academic/Plenum Publishers), pp. 368-391.
reviews
  • 967 Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999).  GroEL-GroES cycling: ATP and nonnative polypeptide direct alternation of folding-active rings.  Cell 97, 325-338.  PubMed   Journal
  • 4853 Shih, W. M., Gryczynski, Z., Lakowicz, J. R., and Spudich, J. A. (2000).  A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin.  Cell 102, 683-694.  PubMed  
  • 4854 Struck, D. K., Hoekstra, D., and Pagano, R. E. (1981).  Use of resonance energy transfer to monitor membrane fusion.  Biochemistry 20, 4093-4099.  PubMed  
  • 4855 Lobb, R. R. and Auld, D. S. (1980).  Stopped-flow radiationless energy transfer kinetics: direct observation of enzyme-substrate complexes at steady state.  Biochemistry 19, 5297-5302.  PubMed  
  • 4856 Lobb, R. R. and Auld, D. S. (1979).  Determination of enzyme mechanisms by radiationless energy transfer kinetics.  Proc. Natl. Acad. Sci. USA 76, 2684-2688.  PubMed  
  • 4857 Bark, S. J. and Hahn, K. M. (2000).  Fluorescent indicators of peptide cleavage in the trafficking compartments of living cells: peptides site-specifically labeled with two dyes.  Methods 20, 429-435.  PubMed   Journal
  • 4859 Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997).  Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.  Nature 388, 882-887.  PubMed   Journal
  • 4861 Van Der Meer, B. W., Coker, G. III, and Simon Chen, S.-Y. (1994). Resonance Energy Transfer: Theory and Data (New York: VCH Publishers, Inc.).

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