FRET Ventures Out of the Lab
Gary Boas, News Editor
Förster resonance energy
transfer (FRET) offers a unique means of visualizing biological activity. The technique
uses two molecules, a donor and an acceptor. Upon excitation, if the molecules are
close to one another, the donor transfers its energy to the acceptor, which then
emits light. This energy transfer occurs only when the molecules are in proximity,
so the emission can reflect, for example, ion binding or protein-protein interactions.
Because of its unique capabilities, FRET contributes
to a wide range of basic science studies: from measurement of conformational changes
to analyte sensing. It also has tremendous potential for biotechnology. Recent studies
have highlighted this potential, exploring its possible uses for drug discovery
and other applications.
At the University of Texas Health Science
Center in Houston, researchers recently developed a FRET method for basic science
application, only to discover that it lends itself to high-throughput screening
for drug discovery. In fact, said Vasanthi Jayaraman, the principal investigator
of the study, the latter is the more important of the potential applications.
Researchers from the University of Texas Health Science Center have devised a FRET-based assay
with a unique donor/acceptor pair to monitor the conformational changes in a certain
glutamate receptor. They discovered that the assay also has strong potential as
a high-throughput screen for drug-discovery applications.
As described in the July 5 issue of
PNAS, she and colleagues Gomathi Ramanoudjame, Mei Du and Kimberly A. Mankiewicz
were interested in the conformational changes in certain glutamate receptors, which
provide an excellent model of changes that occur in its family of proteins. An isolated
ligand-binding domain of the receptor generally exhibits a direct correlation between
a ligand-induced cleft closure and the extent to which it is activated. This is
not always the case, however. A mutation of the receptor exhibits different closures,
suggesting that some ligands or modifications to the receptor can alter the mechanism
of activation.
They chose to explore these conformational
changes with a FRET-based assay, which required very little sample and allowed measurement
of the changes in solution. They tested a number of probes for use with the assay,
eventually hitting upon a pair that Jayaraman describes as a “dream probe.”
This relatively inexpensive, commercially available pair included the maleimide
derivatives of fluorescein for the acceptor and a chelate of terbium for the donor,
and enabled measurements unlike those performed with traditional FRET techniques.
Because the terbium chelate has sharp emission lines, the signal at the acceptor
wavelength did not have any overlap from the donor. Moreover, the acceptor —
in the absence of the donor — had a nanosecond lifetime. As a result, background
signal from the donor or acceptor only could be eliminated.
Johns Hopkins University investigators have developed a directed-assembly
method for surface-supported bilayers. This could contribute to improved FRET monitoring
of membrane proteins, which constitute 90 percent of all drug targets, and thus
advance pharmaceutical applications. Reprinted with permission of Langmuir.
Identification key
Jayaraman noted that identifying this pair was
the key to the study. Once they found it, she said, “it was a breeze.”
They performed the measurements with a cuvette-based fluorescence lifetime spectrometer
made by Photon Technology International of Lawrenceville, N.J. A nitrogen laser
fiber optically coupled to the sample department provided excitation. The fluorescence
emission was collected and passed through a monochromator to a stroboscopic detector
with a photomultiplier tube that detected in the 185- to 680-nm range. The donor-only
lifetimes were collected at 488 nm; the acceptor-only lifetimes, at 515 nm. They
acquired the FRET lifetimes by studying the sensitized emission of the latter.
The experiments confirmed that, in
the mutation of the receptor, the degree of cleft closure is not always a reliable
measure of the extent of activation, suggesting that other factors contribute in
subtle ways. Nonetheless, in all cases, the FRET-based assay clearly distinguished
between closed and activated states. This pointed up its potential as a high-throughput
screen for ligands that are agonists or antagonists of the receptor, especially
because standard plate readers can perform these types of measurements. The receptor’s
agonists are linked to Alzheimer’s and Parkinson’s diseases, for example;
its antagonists may be pharmacologically useful for preventing neuronal degeneration
such as occurs in stroke and amyotrophic lateral sclerosis.
High-throughput screening with this
FRET method can provide important additional information. Typically, binding assays
reveal only whether a drug binds, but this FRET-based assay also can predict how
the drug will function; i.e., will it function as an activator, inhibitor or partial
activator?
The scientists plan to continue their
studies of the conformational changes of the receptors — looking at the full
receptor as opposed to the isolated domain, for example — and intend to explore
use of the technique for high-throughput screening. They would like to develop it
for use with a 96-well plate, as opposed to the cuvette used in the current study.
Also, application of the technique isn’t limited to glutamate receptors; it
can be adapted for use with any number of others.
“We are currently looking at
other types of glutamate receptors as well as the glycine receptor,” mutations
that can lead to startle disease/hyperekplexia, Jayaraman explained.
Pharmacological applications
Membrane proteins — which mediate communication
between a cell and its environment — are crucial to drug discovery. Indeed,
90 percent of all drug targets are membrane proteins. However, there are no good
ways to study them, according to Kalina Hristova, an assistant professor at Johns
Hopkins University in Baltimore. Fluorescence techniques typically involve measurement
of soluble proteins in cuvettes, but membrane proteins are not soluble and reside
in the very complex membrane environment.
In a 2004 Langmuir paper, Hristova
and Edwin Li reported a study in which they successfully quantified interactions
between transmembrane peptides in surface-supported lipid bilayers with FRET imaging,
using a technique they had developed for self-assembly of the bilayers. However,
the peptides in these bilayers were immobile, limiting application of the technique
for biosensing.
In a 2006 Langmuir paper, Hristova
and Li as well as co-authors Mikhail Merzlyakov and Rachel Casas, described an
alternative to the previously reported self-assembled bilayers. With this “directed
assembly” method, the peptides are incorporated into a monolayer as a first
step in the process. Thus, the researchers can control the concentration, orientation
and lateral mobility of the peptides, opening up the potential of the technique,
especially for detecting analytes that affect protein interactions.
In the Aug. 1 issue of the journal,
they reported a study in which they tested the directed-assembly method by imaging
fluorescently labeled proteins in bilayers with FRET. They used a microscope made
by Nikon Corp. of Tokyo, outfitted with a mercury lamp and a SPOT RT camera made
by Diagnostic Instruments Inc. of Sterling Heights, Mich. Excitation and emission
filter cubes for each of the dyes used in the study — Cy3, Cy5, fluorescein
and rhodamine — completed the setup.
Some of the dyes behaved unexpectedly
during the experiments — quenching and disappearing via an unknown mechanism.
“We still have no explanation,” Hristova said. They addressed this by
performing spectral FRET instead, with a fluorometer made by Jobin Yvon of Edison,
N.J. This technique, more commonly used to detect fluorescence in solution, proved
effective for recordings in the directed-assembled bilayers. In fact, it offers
several advantages over imaging: It yields more information and, with a standard
fluorometer such as that used in the study, is relatively inexpensive, at less than
$100,000 for the setup.
Future potential
Furthermore, because the proteins are in surface-supported
bilayers, high-throughput screening is possible. Researchers could perform a number
of measurements on a single chip to begin screening for drugs that interfere with
the membrane proteins. Hristova noted, however, that, although the technology is
amenable to high-throughput measurements, it has not yet been adapted. The investigators
have filed a provisional patent with an eye toward developing it commercially, and
they are exploring whether it is compatible with other classes of membrane proteins
that function differently.
They continue to perform basic science
research with membrane proteins. For example, they are working toward measurement
of the association thermodynamics of complex proteins in membranes. A long-term
goal, Hristova said, is to correlate biological function with the system’s
structural and thermodynamic parameters and to develop a comprehensive model of
signal transduction across the plasma membrane in both health and disease.?
A trend in the past five to 10 years
has researchers working to develop label-free materials that can respond to binding.
Self-amplifying materials such as these could allow for one-step sensing, said Quan
Cheng, an investigator with the University of California, Riverside. A sensor is
dropped into solution and binding occurs, generating fluorescence. This would open
up a variety of applications for high-throughput screening.
Cheng and colleagues recently reported
a “smart sensor” such as this: a polydiacetylene-based vesicle sensor.
Here, binding of analytes to embedded lipid dyes leads to energy transfer, producing
a FRET signal. In the Aug. 1 issue of Langmuir, he and Guangyu Ma took the
work a step further, developing the vesicle as a “mix and detect” fluorescent
sensor for direct detection of bacterial cholera toxin.
Researchers at the University of California, Riverside, have come up with a “mix and detect”
FRET technique for one-step sensing. The method takes advantage of a self-amplifying
material incorporated into a vesicle. The fluorescence is suppressed until a toxin
is introduced, blocking the quenching and effectively switching on the sensor.
The technique offers several advantages
over other methods using self-amplifying materials. First, the materials are easy
to synthesize, as is rarely the case with self-amplifying techniques. Also, operation
is relatively simple and sensitivity relatively high, especially for label-free
detection.
Cheng points to another important aspect
of the technique: the use of “turn on” detection. Most approaches to
self-amplifying detection rely on fluorescence quenching, in which the fluorescence
is present until binding occurs and quenches it. With turn-on detection, in contrast,
the fluorescence is suppressed until binding occurs, essentially switching it on.
This is more attractive, he said, and it provides higher sensitivity.
Turn-on detection
They achieved this by mixing the polydiacetylene
with Bodipy-tagged ganglioside GM1, which leads to substantial quenching of the
self-amplifying material (greater than 60 percent). When the bacterial cholera toxin
is mixed into the solution, it blocks the quenching and effectively turns on the
fluorescence.
They demonstrated the utility of the
sensor by performing measurements with a Jobin Yvon spectrofluorometer. For the
Bodipy-tagged ganglioside GM1, they used a 480-nm excitation wavelength and collected
the emission in the 490- to 600- nm range. The measurements confirmed that the vesicle
allowed for turn-on detection of bacterial cholera toxin. In fact, adding the toxin
even led to a slight increase in the fluorescence signal with respect to the base
line.
Still, Cheng said, there is room for
improvement. “We were able to detect in the nanomolar range, but why can’t
we go for picomolar or even femtomolar? The higher the sensitivity, the better.”
Also, the question has arisen as to whether the technique can be used with more
common antibodies and antigens. “We still need to think about that,”
he added. “If we can apply this strategy [in that way], we can make it a more
generalized form of detection.” This would open up a variety of potential
applications; for example, in medical diagnosis, food safety and perhaps national
security, because the technique can detect bacterial toxins, a potential class of
biowarfare materials.
The researchers are working to patent
the technique and are seeking collaborations within the biotech industry. “There
are only so many things we can do in the lab, in the university setting,”
Cheng concluded. “To make this more applicable requires the involvement of
industry.”
Published: September 2006