New application of imaging technique can change how scientists look at proteins

November 29, 2001

Researchers at the University of Washington have developed a new technique for observing large proteins that gives scientists the most detailed picture yet of the biological workhorses in action and promises to shed light on a wide range of issues, including the biocompatibility of medical implants, blood-clotting processes and how cancer spreads.

"To a large extent, a protein's structure determines its function," said Viola Vogel, associate professor of bioengineering and director of the UW's Center for Nanotechnology. "But, for very large proteins, the precise correlation is poorly defined. Now we have a very efficient way of tracking changes in structure so we can see how it relates to what these large proteins do."

Details of the new application of the technique, known as fluorescence resonance energy transfer, appear in the latest issue of Proceedings of the National Academy of Sciences.

Large proteins are made up of numerous amino acids, strung together in sequence like a strand of pearls. The structure of the protein changes depending on whether those pearls are tightly packed or whether the strand is extended. Along the strand are reactive sites that can bind with other molecules to perform various functions.

Those sites can be buried if the protein folds upon itself, which means they wouldn't be available to bind. Thus, changes in a protein's structure - whether it's compactly folded, unfolded or at some point in between - can change its performance.

Vogel and doctoral students Gretchen Baneyx and Loren Baugh did their initial experiments using fibronectin, a large protein that acts as a sort of super glue to hold cells together. The protein weaves itself into "sticky" attachment sites on cell surfaces and forms thread-like fibrils to connect the cells to one another. Fibronectin plays important roles in embryo development, wound healing and other vital biological functions.

To explore how fibronectin works from an engineering perspective - why it's strung together like a strand of pearls and how structural changes affect function - Vogel's group labeled the protein with fluorescent dyes. The dyes include donors and acceptors, which interact with one another if they are close together. When the donor becomes excited, it releases energy in one of two ways:

So as the protein changes structure, becoming more compact or more stretched out, the distance between the fluorescent molecules changes, which affects the energy transfer. And, as energy transfer changes, so does the color of the light. Red light indicates that the protein is compact and green that it is stretched out. By using a custom-built microscope to keep track of the changes in light, the researchers can precisely track changes in the protein's structure.

"This is allowing us to see structural changes in cell culture that other techniques cannot detect," Vogel said. The next step, which Vogel's group has begun to explore, is to correlate those changes in protein structure with specific functions.

"We're looking to see if we can combine this technique with the use of probes to monitor the exposure of the protein's functional sites, and thus establish structure-function relationships for fibronectin," Baugh said. "We're applying a similar approach to other adhesive proteins whose structure-function relationships have not been resolved," including fibrinogen, which plays an important role in blood clotting and whether the body accepts or rejects medical implants.

Metastasis, or the spread of cancer, is another area that could be better understood through fluorescence resonance energy transfer, Vogel said.

"Some cancer lines are depleted of fibronectin so they are not 'sticky' and migrate much more easily into tissue," she explained. "This technique could give us a better grasp of how that works. Its potential applications are extremely far-reaching. We're just starting to scratch the surface."
-end-
The research was funded by a grant from the National Institutes of Health.

For more information, contact Vogel at 206-543-1776 or vvogel@u.washington.edu.
To view the article on the Web, go to http://www.pnas.org

A high-resolution color image of proteins observed using fluorescence resonance energy transfer is available on the Web at:
http://www.washington.edu/newsroom/news/images/protein1.jpg

The image depicts strands of the protein fibronectin, which binds cells together and is compactly woven on cell surfaces. The protein has been marked with fluorescent dyes. The green filaments are strands that are "stretched out" between cells - they appear green because they are "open," placing fluorescent molecules farther apart, which leads to lower energy transfer among the molecules. The red hues depict fibronectin that is more tightly folded around cell membranes and, because the fluorescent molecules are closer, energy transfer is high, accounting for the reddish color.

University of Washington

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