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MEMBRANE PROTEIN MICROARRAYS
Technology fills gap left by earlier arrays, shows promise for drug screening
CELIA HENRY
Membrane proteins don't function well out of their lipid environment. So pinning them down in microarrays has proven difficult. Now, scientists at Corning have successfully demonstrated microarrays of G-protein-coupled receptors (GPCRs), an important class of membrane-bound proteins [J. Am. Chem. Soc., 124, 2394 (2002)]. Such arrays could be useful for drug screening because many drugs target membrane proteins.
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Fang (left) and Lahiri
CORNING PHOTO |
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Constructing such arrays is "a two-part immobilization problem" involving both the membrane and the protein, says Joydeep Lahiri, group leader of the high-sensitivity assays group in biochemical technologies at Corning. The team also includes senior research scientists Ye Fang and Anthony G. Frutos.
Lahiri and his coworkers developed a system that is mechanically stable but still allows individual molecules to move around in the immobilized membrane. Such lateral fluidity is a property of biological membranes. The surfaces that best balanced those properties were modified with g-aminopropylsilane.
Although they don't have any detailed structural data, the Corning researchers believe that the membranes are immobilized. And because they are housed in the membrane, the researchers think the proteins are immobilized, too. "This membrane protein also stays functional, at least functional as determined by its ability to bind ligands the way it is supposed to," Lahiri says.
After they determined that model lipid systems could be printed on the surface, the scientists turned their attention to real biological systems. They made arrays with combinations of three different families of GPCRs--the adrenergic, neurotensin, and dopamine receptors. The arrays were incubated with fluorescently labeled ligands to screen compounds across different receptor families and within a single family of receptors.
"The affinity of compounds for a particular G-protein-coupled receptor that we find using these arrays is the same as you get by other methods," Lahiri says. This finding "suggests that the GPCR/G-protein complex is preserved in the microspot. It opens the door to the possibility of studying not just ligand binding but ligand agonism," he adds.
"The group at Corning appears to have considerable success in demonstrating function and selectivity of an important class of membrane-associated proteins," says Steven G. Boxer, a Stanford University chemistry professor who has pioneered methods of patterning lipids on surfaces. "The group at Corning, as well as Proteomic Systems and likely other start-ups, is making important progress in this area." Boxer serves as an adviser to Proteomic Systems, a start-up company in Sunnyvale, Calif.
The next step for the Corning group is to fabricate arrays for other types of membrane proteins. "GPCRs are a fairly complex set of proteins," Lahiri says. "If we can work with them, the chances are that we will be able to work with other membrane proteins also."
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| SEEING SPOTS The GPCR microarray (schematic on left) is incubated with fluorescently labeled ligands, both with and without inhibitors. (The pink, green, and red structures under the membrane represent the subunits of the G protein.) In the absence of an inhibitor, the ligand binds to the membrane protein, resulting in a fluorescence signal (top right). In the presence of an inhibitor, no fluorescence is observed (bottom right). |
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