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Rare Earth Elements Excite Protein Probes

Seeing what's going on inside living cells at the molecular level may reveal biological mechanisms and ultimately lead to more effective medicines. While sophisticated microscopes allow scientists to take pictures of a single molecule, capturing images of single molecules in a living cell has been particularly challenging. The molecules must be "tagged" to made visible under the microscope.

Lawrence Miller, assistant professor of chemistry at the University of Illinois at Chicago, hopes to meet that challenge with the help of a four-year, $1.16 million grant from the National Institutes of Health.

"Over the past 10 years, there's been a revolution of sorts in studying protein function in living systems using microscopy to follow dynamic movements and localizations of particular protein molecules," said Miller.

To image a protein, it must be tagged with what is called a reporter -- another protein or even a small organic molecule with special optical properties, such as fluorescence. When fluorescent reporters are illuminated with light of a particular color, they give off a different color light. Fluorescence makes it possible to distinguish reporter-tagged proteins from untagged proteins in the cell.

Common fluorescent reporter molecules make it easy to see multiple copies of a tagged protein in a cell. However, it is difficult to observe a single copy because of other fluorescent molecules in cells. Light from these other fluorescent molecules generates background noise that can obscure the reporter-tagged protein of interest.

But there are ways to distinguish reporter molecules from background fluorescence. All fluorescent molecules have a characteristic lifetime. When a short pulse of light is shined on a molecule, there is a brief delay before fluorescence. The background fluorescence in cells has a lifetime measured in nanoseconds -- billionths of a second.

Miller's lab will build a time-resolved microscope using sophisticated high-shutter-speed cameras to track proteins tagged with a different kind of reporter. The new probes will use lanthanides, the so-called rare-earth elements of the periodic table.

Europium and terbium are particularly promising, Miller said. Their fluorescence is different and more detectable than the commonly used tags.

"They give off multiple colors -- and what's particularly useful, technologically, is that it takes a longer time between when they're excited with a light pulse and the time they fluoresce," he said.

While the whole process happens in a fraction of a second, the lag helps distinguish lanthanide-tagged molecules after the glow of interfering cell fluorescence has faded.

"One purpose of our studies is to demonstrate that we can detect lanthanide reporter-tagged proteins at the single-molecule limit in living cells," said Miller. "That's never been done before."

Lanthanides can also be chemically incorporated into small molecules. Miller's lab aims to synthesize lanthanide reporters that can penetrate cell membranes and bind to proteins of interest with relative ease -- similar to the way drug molecules bind to their targets in cells.

"These tags are like 'smart bombs,'" said Miller. "You add them to cell cultures and they go into cells, find the protein you want to study, and bind with high affinity. It's a straightforward way to selectively label a protein and makes it detectable."

Miller hopes his research will give scientists a better tool to probe protein function within living cells.

Michael Sheetz, professor and chair of biological sciences at Columbia University, will collaborate with Miller by assessing the effects of lanthanide tags and time-resolved microscopy on cell health.

8/26/2008
University of Illinois at Chicago

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