But Fowler and Fields were interested in some of the losers, too, because that would allow them to track the fate of far more variants. So they tried phage display with a new twist: doing it under conditions that increased the chances of binding, so that more phage would make it through each round of selection. They then used high-throughput sequencing to identify and quantify the DNA of the surviving phage, with the DNA counts acting as a proxy for protein function: those sequences increasing in abundance encoded better binders, and those that were depleted encoded faulty ones.

This innovative combination of sequencing and permissive phage display produced a detailed map of how specific amino acids influence the binding ability of a small and well-characterized region of a protein called the human Yes-associated protein 65 WW domain. As reported in the September 2010 issue of Nature Methods, the researchers' approach winnowed more than 600,000 variants of this protein down to 94,606 survivors—a group large enough to reveal how each amino acid position in the protein tolerated mutation. This process pinpointed key positions critical for protein function and showed that both the exact position and the precise amino acid in that position mattered.

"The map you get is a much denser kind of landscape than you would ever get by standard mutagenesis, because with standard mutagenesis you're not able to look at so many variants," Fields says.

		A Rugged RNA-Scape Two miles from the Fields lab, a similar experiment was under way at the Fred Hutchinson Cancer Research Center. Read More

To check how their proxy measure of protein function related to protein structure, they teamed up with David Baker, a UW colleague and HHMI investigator. Using a computational tool developed by Baker called Rosetta, they simulated the stability of their surviving protein variants and found that the relative abundances correlated with the calculated stabilities, with the depleted variants likely to be less stable than the original wild-type protein.

Fields says that because the method is fairly generic, it can be tailored to many biological questions. For example, it could catalog the effects of mutations in disease genes, distinguishing deleterious variants from harmless ones. In drug development, the method could assay the range of protein variants vulnerable to a drug—something important to know for diseases such as cancer, in which a runaway mutation can allow proteins to develop drug resistance.

It can even provide a reality check for proteins designed by computers and humans, says Baker. "A brand new protein is born out of nowhere, you know nothing about it. This technique offers the potential to immediately know what the role of every amino acid is."

And the time it took to get from a frustrating group meeting to publication of a new method? One year. "It was one of these magical experiences in science that doesn't happen very often," Fowler says.

PAGE 1 2		Go Back