Protein lineages: randomness was crucial to ancient genetic changes.

After resurrecting a protein from an animal species that lived about 470 million years ago, a team of scientists has now partly reconstructed the protein's evolutionary history.

The rare glimpse into a protein's past reveals how a sequence of mutations caused the ancestral molecule to acquire a function possessed by modern forms of the protein, which is present in people and other vertebrates.

The research addresses a long-standing debate among biologists: If the evolutionary clock were turned back and allowed to run again, would the pressures of natural selection steer an organism to the same outcome, or would chance mutations produce a different result?

"What we observed suggests that there's a significant degree of contingency and randomness in evolution," says research team leader Joseph W. Thornton of the University of Oregon in Eugene.

In previous work, Thornton's team inferred the ancient protein's genetic code by comparing many species' genes for glucocorticoid receptors, which are the modern descendants of the protein. If a portion of the gene is identical in several species, chances are that those species all inherited that portion from a common ancestor. This kind of analysis enabled Thornton and his colleagues to reconstruct the ancestral gene with about 99 percent confidence.

In the current study, the team synthesized the ancient gene and inserted it into cells grown in the lab. The cells translated the gene into its protein, whose three-dimensional structure the researchers then determined. They also inferred the genetic code of descendants of the protein dated to roughly 440 million and 420 million years ago and estimated their 3-D structures. The scientists found that sometime during that 20-million-year time span, the protein developed the ability to bind exclusively to cortisol, a stress hormone.

By looking at how the protein's 3-D structure changed during that time, the team identified three crucial mutations that appeared to be responsible for the protein's ability to bind cortisol. But when the researchers applied those mutations to the 440-million-year-old version, structural weaknesses within the protein interfered with the mutations' effects, and the protein couldn't bind cortisol at all.

The scientists then found additional mutations that had no effect on the protein's function but that buttressed the protein's weak spots. Applying these functionally neutral mutations, as well as the key mutations, produced a protein that, unlike its older version, bound only cortisol. The team's findings appear online and in an upcoming Science.

Thornton and his colleagues reasoned that the neutral mutations must have occurred first, paving the way for the function-causing mutations. But because natural selection can act only on changes that make a functional difference, the earlier, enabling mutations were essentially invisible to its steering influence.

A different set of random, neutral mutations might have buttressed the protein in ways that would have facilitated a different key mutation. This would have led to an alternate "evolutionary road not taken," Thornton speculates.

If neutral, enabling mutation prove to be common in protein evolution, "that's pretty groundbreaking," comments Christopher C. Dascher of the Mount Sinai School of Medicine in New York.