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TECHNIQUES
Fluorescence resonance energy transfer (FRET)
by Hays Rye
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Overview
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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
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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.
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Can be applied to any molecular system for which a fluorescent derivative can be produced.
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Measures
molecular distances (15-100 Å) that are similar to the dimensions of
many biological macromolecules and are difficult to study with other
methods.
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Is capable of providing both proximity and kinetic information over a wide range of time scales (nanoseconds to hours).
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Through
the use of fluorescent proteins like GFP, FRET can be readily applied
to the study of molecular interactions inside living cells.
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Easily adapted to a wide variety of instrument formats.
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Controls, considerations, and disadvantages
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Accurate distance measurement is generally limited to a single pair of sites in any given experiment.
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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.
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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.
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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.
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Applications
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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.
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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).
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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.
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Credits:
Images courtesy of Rex Chisholm.
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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).
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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.
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Protein-nucleic acid interactions and nucleic acid hybridization.
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Protein and nucleic acid folding.
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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).
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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).
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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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4848 Stryer, L.
(1978).
Fluorescence energy transfer as a spectroscopic ruler.
Annu. Rev. Biochem. 47, 819-846.
PubMed Journal
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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
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4850 Wu, P. and Brand, L.
(1994).
Resonance energy transfer: methods and applications.
Anal. Biochem. 218, 1-13.
PubMed Journal
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4851 Clegg, R. M.
(1995).
Fluorescence resonance energy transfer.
Curr. Opin. Biotechnol. 6, 103-110.
PubMed
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4852 Miyawaki, A.
(2003).
Visualization of the spatial and temporal dynamics of intracellular signaling.
Dev. Cell 4, 295-305.
PubMed Journal
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4858 Tsien, R. Y.
(1998).
The green fluorescent protein.
Annu. Rev. Biochem. 67, 509-544.
PubMed Journal
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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.
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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
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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
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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
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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
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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
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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
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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
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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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© Jones and Bartlett Publishers (2007)
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