The Trouble with Membranes

Membrane proteins comprise the majority of drug targets.

Aileen Constans
Dec 5, 2004
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Courtesy of Steve Zweig

Membrane proteins comprise the majority of drug targets. So naturally, the protein biochip industry is keen to array them. But membrane protein arrays have been difficult to build, because these proteins are notoriously tough to stabilize. Now a group of investigators is working to overcome this problem.

Steve Zweig of Molecular Pathways, which recently patented a technique for studying drug candidate binding to G protein-coupled receptors (GPCRs) in an array format,1 says previous attempts to make membrane protein microarrays failed because of steric problems. With the membranes positioned only nanometers from the array substrate surface, the receptors' cytoplasmic domains (which protrude below the lipid bilayer) were distorted and rendered inert.

Membrane proteins are laterally mobile within the membrane, says Zweig, so any microarray technology has to allow for such movement. "Because the membrane receptors were sort of moving parallel to the support, there really wasn't...

A GROWING FIELD

Steven Boxer of Stanford University points out that Zweig's invention is not technically a membrane protein microarray but rather a binding assay composed of surface-bound vesicles. Boxer, a chemist, has designed and published descriptions of arrays of mobile lipid vesicles tethered to a lipid bilayer substrate by 24-mer oligonucleotides spotted onto a glass slide.2

Individual lipid vesicles will spontaneously assemble into a single membrane bilayer on several types of surfaces, including silica, but array developers have had difficulty attaching membrane-associated proteins to these membranes. Membrane proteins can, however, easily be incorporated in individual vesicles. Boxer and colleagues used vesicles containing short oligos to create a lipid membrane layer and flowed loose vesicles containing antisense sequences over the membrane; these vesicles became tethered to the membrane when they came into contact with a complementary sequence. Boxer is currently developing protein-containing vesicle arrays based on this technique.

A team of scientists at Corning has already developed and is in the process of testing its own membrane-protein microarrays. Corning researcher Ye Fang and colleagues printed spots containing GPCRs and associated lipids onto a substrate; the arrayed proteins were then screened for binding to fluorescently labeled ligands.3

Fang explains that his group aimed to accomplish three tasks: overcome the low stability of lipid membranes in air, preserve the lateral fluidity of lipid membranes, and maintain the native conformations of GPCRs immobilized on the arrays. After screening more than 20 surface chemistries, Fang and colleagues found γ-aminopropylsilane, which had the necessary properties.

Other competing technologies, including that of Zweig and Boxer, are only water-stable, but Fang's arrays are air-stable, which facilitates storage. The arrays also are easy to fabricate and support multiple types of receptors. Says Fang: " [γ-aminopropylsilane] preserves the lateral fluidity of biological membranes, and is important for the biological function of membranes associated with it."

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