Tiny Tools That Glow

Each summer the waters of Puget Sound whirl with the choreography of an underwater ballet. It is a mating dance that is as ancient as it is beautiful. Scores of Aequorea victoria, a diaphanous jellyfish, gather near the surface to release their precious cargo of eggs and sperm.

In the cold, dark waters of the North Pacific, the jellyfish are nearly transparent. When physically disturbed, however, a circlet of tiny green lights appears on the rim of the animals’ bell-shaped bodies. Each one marks the location of a tiny light organ.

In 1962, researchers isolated the molecule known as green fluorescent protein (GFP). It is GFP that causes A. victoria to glow green. By the 1990s, scientists had genetically engineered GFP to shed light on another kind of choreography—the chemical traffic taking place inside a living cell.

“GFP vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology,” wrote Roger Tsien of the Howard Hughes Medical Institute in Washington, D.C.

Few scientists are as enthusiastic about the potential of GFP as Rebekka Wachter, an assistant professor in department of chemistry and biochemistry at Arizona State University. She says that GFP and a group of related proteins that are responsible for bioluminescence in sea corals have become indispensable tools. Scientists use them to study diseases ranging from cancer and developmental disabilities to genetic defects. By developing mutant forms of GFP, Wachter and her colleagues hope to expand the toolbox of practical techniques that are available to biomedical researchers.

GFP provides researchers with an elegantly simple way to peer inside the workings of living cells. Like other proteins, GFP is composed of a particular sequence of amino acids—in this case, serine, tyrosine and glycine. Wachter explains that these amino acids are arranged on a chain like beads on a string. But during a series of chemical transformations, the protein chain folds into a barrel shape. The amino acids are buried deep within the interior. Together they form what is known as a chromophore. The chromophore is the part of GFP that emits the protein’s signature green light.

Researchers can track the traffic within a cell. They simply attach the genetic coding for GFP to the DNA of the cellular component they’re interested in studying. The chemical signals that activate these cellular components also trigger the generation of GFP, causing them to fluoresce green.

Scientists use sophisticated fluorescence microscopes to see the color. As a result, they can get real-time glimpses into cellular activity. This technique has been used in the laboratory to document the spread of viruses. It has also been used to view the development of nerve cells in the nematode Caenorhabditis elegans.

Wachter says that GFP also has proven useful as a biosensor that can deliver precise measurements of cellular conditions. For example, the ASU researcher has helped to develop a GFP variant that can detect salt concentrations in cells. Such monitoring is important to laboratory scientists studying the fatal disease cystic fibrosis.

Researchers have noted salt imbalances in the cells of patients suffering from the disease. The GFP signal allows them to better understand the breakdown in a regulator that is responsible for the transport of chloride ions across the cell membrane.

Another GFP variant that she has helped to design can signal the pH—the acidity or alkalinity—within the cell environment.

Using GFP in this manner, Wachter says, allows researchers to study cellular processes without resorting to invasive methods such as inserting needles or introducing enzymes that could disrupt the cell’s normal biological processes. The only ingredient needed for jumpstarting the protein-maturation process is atmospheric oxygen.

GFP does have its drawbacks as a research tool—limitations that Wachter is trying to overcome. For example, GFP is a relatively large molecule. That rules out its use in situations where smaller, more nimble molecules are needed. And it can take up to an hour for the protein to fold and produce a chromophore. That time lag is far too long for tracking many cellular processes.

Wachter’s group is working to resolve these issues by answering some fundamental questions about GFP’s physical chemistry. Why, for example, is GFP green when proteins from sea corals produce chromophores whose colors range from blue and yellow to red?

Wachter also would like to fill in some of the gaps in the understanding of how GFP folds into its barrel shape and produces its fluorescent-green chromophore. Answering these questions could help find a way to speed up the process of chromophore formation. It might also allow scientists to transplant GFP chemistry into an unrelated but much smaller protein, findings that could open up whole new areas of biomedical research.

Such major scientific breakthroughs, however, don’t come without grit and tenacity. They also take time.

“Collecting enough information on these colored proteins so that you can actually design changes in the structure could be a few weeks to many months to a couple of years,” Wachter says.

The protein must first be purified and then made to crystallize. The highly ordered, but fragile, protein crystals are then subjected to high-energy X-rays. The process is known as X-ray crystallography. Scientists use it to reveal a protein’s three-dimensional structure.

Each GFP molecule contains some 2,000 visible atoms. Another several thousand hydrogen atoms are too light to show up on an X-ray image. It is only after this detailed information has been processed and analyzed that Wachter can tinker with GFP’s redesign. Mutated proteins that result in desired functions, such as faster color acquisition, are then subjected to a whole new round of changes.

“Success hinges on being able to stick with something and not walk away the first time you’re frustrated,” Wachter says with a smile. “Once you get results—any kind of results—it’s a huge amount of fun.”—Adelheid Fischer