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  • FRET Ventures Out of the Lab

Sep 2006
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.”

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